WO2012031115A2 - In vivo anti-tumor activity of a recombinant il-7/hgf(beta) hybrid cytokine and associated methods of use - Google Patents

Abstract

The present invention relates to a hybrid cytokine comprising IL-7 and HGF-β and/or bioactive portions thereof, including a chimeric or fusion protein comprising an IL-7 polypeptide or bioactive portion thereof, and an HGF-β polypeptide or bioactive portion thereof, joined contiguously in a single polypeptide chain; and wherein the hybrid cytokine polypeptide inhibits the growth, proliferation, and/or metastasis of a cancer cell, in vivo.

Description

IN VIVO ANTI-TUMOR ACTIVITY OF A RECOMBINANT IL-7/HGF HYBRID

CYTOKINE AND ASSOCIATED METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority to U.S. Provisional Patent

Application Serial No. 61/379, 101 filed on 01 September 2010, which is incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[002] The present application hereby incorporates by reference, in its entirety, the

Sequence Listing filed in electronic form herewith, file name: 98121.00197SEQLIST_ST25.txt, size: 28 KB, made using Patentln 3.5 software on 25 August 201 1.

FIELD OF THE INVENTION

[003] The present invention relates to the discovery of a novel recombinant hybrid cytokine comprising IL-7 and HGF-beta (herein, "IL-7/HGF-P"), methods for its production, and therapeutic use in vivo, e.g., as an anti-tumor agent.

BACKGROUND

[004] Hepatocyte growth factor (HGF), also called scatter factor SF, is a heparin- binding glycoprotein that is secreted as a biologically inert single chain (pro-HGF) and is converted to its bioactive form by targeted protease digestion to an active disulfide-linked heterodimer. HGF is a natural ligand for the c-MET proto-oncogene product of a novel family of heterodimeric receptor tyrosine kinases that include Ron, Sea and Sex. It is a pleiotropic factor derived from the mesenchyme that regulates epithelial, neural, endothelial, muscle and hemopoietic cell growth, motility, morphogenesis and regeneration in many tissues and organs. The importance of HGF is seen in transgenic mice homozygous for a null mutation in the HGF gene. Such mice do not survive beyond fifteen days of embryonic development.

[005] Mature bioactive HFG is a heterodimer consisting of a 60 kD alpha and 30 kD beta chain held together by a single disulfide bond. Structure function analysis indicates that the beta chain of HGF is required for mitogenic activity, whereas the receptor-binding domain is located in the alpha chain. Its primary structure is highly conserved among mouse, rat, human and other species. The alpha chain contains a hairpin loop at its amino terminus and four unique domains known as "kringles", while its beta chain contains a serine protease-like structure. Hence, HGF is closely homologous to plasminogen, but has no known protease activity due to mutation of the catalytic site.

[006] HGF has been reported to be sequestered in the extracellular matrix (ECM) in vitro as well as in vivo, where it is bound to cell surface heparin sulfate glycosoaminoglycans. In general, HGF mRNA is expressed in stromal cells, whereas HGF receptor expression is mainly detected in epithelial and other parenchymal cells. This pattern suggests that HGF is an important paracrine mediator of the interaction between the parenchymal and stromal components of various tissues both during fetal development and in the maintenance of homeostasis in adult tissues.

[007] Although a great deal is known about the actions of HGF in nonhemopoietic tissues, the role of HGF in the regulation of hematopoiesis, particularly lymphopoiesis, is fragmentary. HGF has been proposed to regulate hematopoiesis in mouse fetal liver and adult bone marrow in vivo, where it apparently can substitute for the stem cell factor (SCF) and c-kit system. HGF is produced by bone marrow (BM) stromal cells and synergizes with IL-3 or GMCSF to support the growth of hemopoietic progenitor cells (HPCs) and myeloid tumor cell lines, all of which express the HGF receptor, c-MET. In the presence of erythropoietin, HGF induces the formation of colonies along the erythroid lineage, whereas in the presence of erythropoietin plus SCF, HGF supports the growth of multipotent colonies. Similarly, upregulation of the HGF receptor on primitive hematopoietic cells may be induced by IL-11 ; and the synergistic interaction of these two cytokines may result in i vitro colony formation by hemopoietic stem cells (HSCs). However, HGF alone does not appear to stimulate proliferation of hemopoietic precursors. The latter may be attributed to enhancement by HGF of signal transduction by lineage-specific cytokines.

[008] HGF has been found to promote adhesion of HPCs to fibrinectin in vitro, and may be involved in a novel paracrine signaling pathway regulating integrin-mediated adhesion and migration of B cells in germinal centers. Messenger-RNA for c-MET has been identified in thymocytes as well as in early B-lineage cells in bone marrow. It is hypothesized that HGF may be involved in a novel paracrine signaling pathway that regulates integrin-mediated adhesion and migration of B-cells in germinal centers. Thus, HGF may be one of the long sought mediators of paracrine interactions between stromal and lymphohematopoietic cells. Furthermore, HGF seems to preferentially affect hematopoietic cells in a window of differentiation between multipotent progenitors and committed precursors. For example, the addition of HGF to fetal thymus organ cultures is known to increase the generation of mature T cells.

[009] Interleukins are a class of proteins that induce growth and differentiation of lymphocytes and hematopoetic stem cells. One interleukin in particular, IL-7, has been demonstrated over the past decade to have an essential role in the development and differentiation of murine pre-B cells. IL-7 is essential for the development, maintenance and regeneration of B and T cells, and is also an activation factor for monocytes, macrophages, DCs and natural killer (NK) cells (Fry TJ, Mackall CL. Interleukin-7: from bench to clinic. Blood 2002; 99:3892-904).

[0010] We previously purified a novel hybrid cytokine consisting of IL-7 and HGF from mouse bone marrow (BM) stromal cells in a unique long-term BM culture system that selectively generated TdT⁺ B-lineage precursors (McKenna S, Chen F, Lai L Goldschneider I. Identification of an IL-7-associated pre-pro-B cell growth-stimulating factor (PPBSF) I. Production of the non-IL-7 component by bone marrow stromal cells from IL-7 gene-deleted mice. J Immunol 1998;160:2272-9; Lai L, Chen F, McKenna S, Goldschneider I. Identification of an IL-7-associated pre-pro-B cell growth-stimulating factor (PPBSF). II. PPBSF is a covalently linked heterodimer of IL-7 and a Mr 30,000 co-factor. J Immunol 1998;160:2280-6; Lai L. and Goldschneider I. Cutting Edge: Identification of a hybrid cytokine consisting of Interleukin-7 and the β-chain of the Heptocyte Growth Factor/Scatter Factor. J Immunol 2001 ;167:3550-4). Consequently, the formation of a rIL-7/HGF-P hybrid cytokine appears designed to regulate lymphohematopoietic stem and progenitor cell development.

[0011] We have cloned and constructed a recombinant IL-7/HGF- hybrid cytokine gene, in which IL-7 and HGF-β cDNAs are joined in a single open reading frame (ORF) and connected by a flexible linker. We have expressed the gene to produce a single-chain or chimeric recombinant IL-7/HGF-P hybrid cytokine protein that stimulates the growth of day 12 spleen colony-forming units, common lymphoid progenitors, early B-lineage cells and thymocytes in vitro. The rIL-7/HGF-P cross-links and induces juxtacrine interactions between the IL-7 and HGF (c-Met) receptors on the cell surface. This in turn results in signal "cross-talk", the appearance of novel phosphorylated proteins downstream, and functional readouts that are not induced by non-complexed IL-7 and/or HGF-β (Lai L., Zeff, RA, and Goldschneider I. A recombinant single-chain IL-7/HGF hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-Si₂, CLPs, and pre-pro-B cells. Blood 2006;107:1776-84).

[0012] Despite the functions of IL-7 and HGF on normal cell growth and development,

IL-7 and HGF differ fundamentally with respect to their effects on cancer cells. In several animal models of neoplasia, IL-7 has been shown to have antitumor effects mediated by CD4 and/or CD8 T cells (Hock H, Dorsch M, Diamantstein T, Blankenstein T. Interleukin 7 induces CD4 T cell-dependent tumor rejection. J Exp Med 1991 ;74:1291-8; Aoki T, Tashiro K, Miyatake S, et al. Expression of murine interleukin 7 in a murine glioma cell line results in reduced tumorigenicity in vivo. Proc Natl Acad Sci U S A 1992;89:3850-4; Murphy WJ, Back TC, Conlon KC, et al. Antitumor effects of interleukin-7 and adoptive immunotherapy on human colon carcinoma xenografts. J Clin Invest 1993;92: 1918-24; Andersson A, Yang SC, Huang M, et al. IL-7 promotes CXCR3 ligand-dependent T cell antitumor reactivity in lung cancer. J Immunol 2009;182:6951-8; McBride WH, Thacker JD, Comora S, et al. Genetic modification of a murine fibrosarcoma to produce interleukin 7 stimulates host cell infiltration and tumor immunity. Cancer Res 1992; 52:3931-7; Li B, VanRoey MJ, Jooss K. Recombinant IL-7 enhances the potency of GM-CSF-secreting tumor cell immunotherapy. Clin Immunol 2007;123: 155-65; Pellegrini M, Calzascia T, Elford AR, et al. Adjuvant IL-7 antagonizes multiple cellular and molecular inhibitory networks to enhance immunotherapies. Nat Med 2009;15:528-36), whereas HGF supported the growth, invasion and metastasis of many types of cancers (Birchmeier, C. et al. Met, metastasis, motility and more. Nat Rev Mol Cell Biol 2003; 4: 915-25).

[0013] Given the potential therapeutic use of rIL-7/HGF-P to enhance B- and T-cell reconstitution in immunocompromised patients, especially following BM reconstitution (Lai L., Zeff, RA, and Goldschneider I. A recombinant single-chain IL-7/HGF- hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-Si₂, CLPs, and pre-pro-B cells. Blood 2006;107:1776-8), it was important to determine whether rIL-7/HGFp positively or negatively affects tumor cell growth in vivo. SUMMARY

[0014] As presently described, it was surprisingly and unexpectedly discovered that the intratumoral injection of an IL-7/HGF- hybrid cytokine (herein, "IL-7/HGF-P") significantly inhibits local tumor growth. In addition, the systemic administration of IL-7/HGF-P significantly inihibits the formation of tumor metastases. Without being limited to any particular theory, it appears that the beneficial effects are mediated by the expansion and activation of tumor-infiltrating T cells (TILs) and Dendritic Cells (DCs). Therefore, described herein are nucleic acids encoding an IL-7/HGF-P hybrid cytokine, the cognate polypeptides, compositions comprising the same, including therapeutic compositions including an excipient, methods of use and manufacture of the same.

[0015] Accordingly, in one aspect, the description provides a hybrid cytokine polypeptide complex comprising IL-7 or a bioactive portion thereof, and HGF-β or a bioactive portion thereof. In certain embodiments, the polypeptide components are bound by non-covalent interactions. In other embodiments, the polypeptide components in the complex are bound covalently. In still additional embodiments, the polypeptide components in the complex are covalently bound to at least one other moiety as described herein.

[0016] In an additional aspect the description provides a hybrid cytokine polypeptide that is a chimeric protein comprising IL-7 or a bioactive portion or component thereof, joined in a single, contiguous polypeptide chain with HGF-β or a bioactive portion or component thereof.

[0017] In additional aspects, the polypeptides provided by the invention comprise biologically-active (i.e., bioactive) portions and/or variants of the IL-7 and/or HGF-β components. Such variants include any homologs, orthologs, paralogs, and homologous peptides to either IL-7 (SEQ ID NO:7, 8) or HGF-β (SEQ ID NO:3, 4, 13, and 14; See FIGs. 37 and 38). For example, variants encompassed by the invention include substitution analogs wherein one or more amino acids have been substituted with different amino acids, deletion analogs wherein one or more amino acids have been deleted, and addition analogs wherein one or more amino acids have been added. Preferably, the substitution, deletion, or addition is made at a residue that does not diminish the beneficial biological activity of the complex or chimeric protein.

[0018] Deletions and additions of one or more amino acids are made either within an internal region of the polypeptide or at the amino or carboxyl terminal ends. Additional potential variations include other heterodimeric (or multimeric) cytokine complexes containing IL-7 and/or HGF (alpha and/or beta chains), and other hybrid cytokines unrelated to either IL-7 or HGF, whether naturally occurring or artificially created, including those that bind to the receptors for HGF, IL-7, and/or yc.

[0019] In additional aspects, the description provides IL-7/HGF- hybrid cytokine polypeptide complexes or chimeras that are also complexed, bound, or conjugated to one or more chemical moieties to improve and/or modify, for example, bioavailability, half-life, efficacy, and/or targeting. In certain aspects of this embodiment, the IL-7/HGF-P hybrid cytokine polypeptide is complexed or bound, either covalently or non-covalently with, for example, cationic molecules, salts or ions, lipids, glycerides, carbohydrates, amino acids, peptides, proteins, other chemical compounds, for example, phenolic compounds, and combinations thereof. In certain aspects, the description provides a complexed and/or chimeric IL-7/HGF-P polypeptides conjugated to another polypeptide, for example, an antibody. In certain embodiments the antibody is specific for a protein target at or near the receptor complex for the IL-7/HGF-beta hybrid molecule.

[0020] In another aspect, the description provides a nucleic acid encoding one or more components of a hybrid cytokine of the invention. In certain embodiments, the description provides a nucleic acid encoding within a single open-reading frame, an IL-7 (SEQ ID NO:7, 8, 10, and 12) polypeptide or active portion thereof contiguous with an HGF -beta polypeptide (SEQ ID NO:3, 4, 13, and 14) or active portion thereof (i.e., a chimeric IL-7/HGF-P hybrid cytokine or IL-7/HGF"P hybrid cytokine fusion protein). In another aspect, the chimeric IL-7/HGF-beta nucleic acid further comprises a polynucleotide linker sequence encoding from 1 to 100 amino acids, which is disposed between the IL-7 encoding polynucleotide (SEQ ID NO: 10, 12) and the HGF-beta encoding polynucleotide (See SEQ ID NO: l 1 , 12). In any of the embodiments of the chimeric nucleic acid of the invention, the polynucleotide encoding IL-7 can be positioned 5' of the HGF-beta polynucleotide, and/or the linker polynucleotide. In an alternative embodiment, the HGF-beta polynucleotide is positioned 5' of the IL-7 polynucleotide, and/or the linker polynucleotide.

[0021] In other aspects the description provides a nucleic acid vector, plasmid or artificial chromosome comprising a nucleic acid encoding a single-chain or chimeric IL-7/HGF- β hybrid cytokine for its convenient cloning, amplification, transcription, and/or translation. In still other aspects the nucleic acid encoding a IL-7/HGF-P hybrid cytokine of the invention is operably linked to one or more transcription regulatory nucleic acid sequences. The vector or plasmid nucleic acids may be stably integrated into the host cell's genome or maintained episomally.

[0022] In a another aspect, the vector, plasmid or artificial chromosome is suitable for expression in a prokaryotic or eukaryotic cell; for example, an insect cell, a mammalian cell, a plant cell, or a bacterial cell. In certain embodiments, the description provides a host cell comprising the chimeric IL-7/HGF-P hybrid cytokine nucleic acid sequence as described herein. In certain aspects, the host cell further comprises a vector or plasmid nucleic acid containing one or more transcription regulatory nucleic acid sequences operably linked with the chimeric IL- 7/HGF-beta nucleic acid sequence of the invention. In certain embodiments, a nucleic acid vector or plasmid containing a chimeric IL-7/HGF-p hybrid cytokine nucleic acid is transformed or transfected into the host cell allowing for transient, stable or inducible expression of the chimeric IL-7/HGF-P polypeptide.

[0023] In another aspect, the description provides a host cell that has been modified to overexpress IL-7, HGF or both. In another aspect, the description provides a host cell overexpressing IL-7 and/or HGF-β protein in which an exogenous promoter or enhancer has been inserted via homologous recombination into the target gene operon to result in increased or inducible protein expression. In other aspects, the endogenous, i.e., naturally occurring, target gene promoter or enhancer is modified or mutated to result in enhanced or inducible expression of chimeric IL-7/HGF-P polypeptide.

[0024] In another aspect, the description provides an antibody which binds specifically to an epitope of a chimeric IL-7/HGF-P polypeptide. In certain embodiments, the antibody is an anti-idiotypic antibody capable of binding immunospecifically to the heterocomplex receptor and mimic the activity of the protein complex for use as a therapeutic alone or in combination with the chimeric cytokine described herein.

[0025] In a related aspect, the description provides a method for producing an IL-7/HGF- β hybrid cytokine protein. In certain embodiments, the process includes providing an IL-7 encoding polynucleotide (SEQ ID NO: 10, 12), and an HGF-β encoding polynucleotide; linking, in a single contiguous polynucleotide chain; the IL-7 and HGF-β nucleic acids being in a single continuous open-reading frame; inserting the chimeric or fusion nucleic acid construct into a vector or plasmid; transforming the vector or plasmid into a suitable host cell capable of expressing the chimeric polypeptide. In certain embodiments, the method also includes the addition of at least one other polynucleotide contiguous and in a single continuous open-reading frame with the chimeric IL-7/HGF-P nucleic acid, which results in the expression of a fusion protein. The additional polynucleotide can be linked on the 5' or 3' end of the chimeric cytokine or in between the IL-7 and HGF-β encoding polynucleotides. Fusion protein constructs contemplated by the present invention include, for example, peptide moieties useful for protein isolation and purification such as GST, fluorescent proteins, multiple histidine residues, antibody epitope tags, a cell sorting signal sequence, and the like. Other fusion constructs may include nucleic acid sequences encoding one or more amino acids that links the two polypeptides and reduces steric or allosteric hindrance.

[0026] In certain aspects, the chimeric IL-7/HGF-p polypeptide as described herein comprises at least one modified amino acid. Amino acid modifications contemplated by the present invention include, for example, phosphorylation, acetylation, methylation; indolizidinone amino acids, D-amino acids, amino acid mimetics, amino acid analogs, etc.

[0027] In other aspects, described herein are methods of inducing the proliferation and/or activation and/or infiltration, in vivo, of T lymphocytes and/or Dendritic cells comprising administration of a composition comprising an effective amount of an IL-7/HGF-P hybrid cytokine to an individual, wherein the hybrid cytokine is effective at inducing the proliferation and/or activation and/or infiltration, in vivo, of T lymphocytes and/or Dendritic cells.

[0028] In other aspects, the description provides methods for treating and/or preventing a disease or condition, in vivo, in an individual related to the detrimental effects of improper cell proliferation and/or differentiation. In certain embodiments, the methods of the invention comprise administering an effective amount of an IL-7/HGF-P hybrid cytokine in a pharmaceutically acceptable form to an individual in need thereof. In certain embodiments, the IL-7/HGF"P hybrid cytokine provided by the invention is administered together with a pharmaceutically acceptable carrier, excipient, adjuvant, amino acid, peptide, polypeptide, chemical compound, drug, biologically active agent or a combination thereof.

[0029] As such, in another aspect the description provides therapeutic compositions comprising a IL-7/HGF- hybrid cytokine in a pharmaceutically acceptable form together with at least one pharmaceutically acceptable carrier, excipient, adjuvant, amino acid, peptide, polypeptide, chemical compound, drug, biologically active agent or a combination thereof. [0030] In any of the therapeutic methods provided described herein, the hybrid cytokine can be administered by any pharmaceutically suitable route recognized by those of skill in the art, for example, enteral, intravenous, intra-arterial, parenteral, topical, transdermal, nasal, and the like. In addition, the therapeutic may be in any pharmaceutically acceptable form such as, for example, a liquid, lyophilized powder, gel, pill, controlled release capsule, and the like, which is now known or becomes known to those of skill in the art.

[0031] In still another aspect, description provides therapeutic compositions comprising an effective amount of a hybrid cytokine provided by the invention in combination with an effective amount of at least one other biologically active agent, e.g., an anti-oncogenic agent, anti-angiogenic agent, or the like. Accordingly, the description also provides methods for inhibiting cancer cell growth and/or metastasis, in vivo, comprising administering a composition comprising an effective amount of a hybrid cytokine as described by the invention together with at least one other biolocially active agent, and a pharmaceutically acceptable carrier, wherein the composition is effective in inhibiting the growth, proliferation, and/or metastasis of a cancer cell.

[0032] Additional advantageous features and functionalities associated with the compositions, methods, and processes of the present invention will be apparent from the drawings presented herein, as well as the detailed description which follows. The publications and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are listed in the appended bibliography.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying figures, which are incorporated in and constitute part of the specification, illustrate presently preferred embodiments of the invention, and together with the general description given above and the detailed description of the preferred embodiments given below, serve to explain the principles of the invention.

[0034] FIG. 1 provides bar graphs of the number of pre-pro-B and pro-B cells versus pre-

B cells derived from rat bone marrow lymphoid cells grown in a pro-B type culture as described (FIGS. 1A and 1C) and pre-B type culture (Whitlock/Witte) (FIGS. IB and ID) over a twenty- one day period emanating from either freshly-harvested cells in primary culture (FIGS. 1A and IB) or emanating from day 10 culture-generated rat bone marrow lymphoid cells in secondary culture (FIGS. IC and ID).

[0035] FIG. 2 is a graph of the sequential proliferation and differentiation of four subsets of B-cell progenitors, pre-pro-B (TdT), pre-pro-B (TdT⁺), pro-B (TdT⁺) and pro-B (TdT), cultured in the pro-B culture system as described, over ten (10) days of culture.

[0036] FIG. 3 is a graph illustrating the relative increase in total pre-pro-B cells freshly harvested from rat bone marrow cultured over an eight (8) day period.

[0037] FIG. 4 is a bar graph of growth of rat bone marrow lymphoid cells in different media, total as well as those displaying the TdT marker.

[0038] FIGS. 5 provides bar graphs of the number of cells (total, pre-pro-B, pro-B, and pre-B) found in primary culture (FIG. 5A) and secondary culture (FIG. 5B) given different combinations of normal medium (NM), bone marrow stromal conditioned medium (CM), and/or rIL-7.

[0039] FIG. 6 is a bar graph of the number of cells (total, pre-pro-B, pro-B, and pre-B) found in a IL-7 (+/+) conditioned medium (CM) versus eluate.

[0040] FIG. 7 is a western immunoblot of PPBSF developed with anti-IL-7 monoclonal antibody in pre-B and pro-B cell conditioned medium under reducing and non-reducing conditions.

[0041] FIG. 8 is a western immunoblot of PPBSF developed with anti-IL-7 monoclonal antibody in designated cell medium with and without rIL-7 (SEQ ID NO:7, 8).

[0042] FIG. 9 is a western immunoblot of PPBSF developed with anti-IL-7 monoclonal antibody in designated combinations of conditioned medium.

[0043] FIG. 10 is a western immunoblot of PPBSF developed with antiserum to PPBSF and adsorbed with IL-7 (SEQ ID NO: 7, 8) for designated conditioned medium, under reducing and non-reducing conditions.

[0044] FIG. 1 1 is a western immunoblot electrophoresed under reducing and non- reducing conditions demonstrating a 30 kD molecule as the non-IL-7 component of PPBSF in (+/+) CM Pro-B.

[0045] FIG. 12 is a bar graph of the number of B220+ lymphoid cells (total, pre-pro-B, pre-B, pro-B) plus and minus anti-PPBSF-coF monoclonal antibody. [0046] FIG. 13 is a partial NH2-terminal amino acid sequence identity of purified mouse

PPBSF cofactor, and its comparison with the published sequence for the HGF-beta chain in mouse (SEQ ID NO:3, 4).

[0047] FIG. 14 is a bar graph of HGF-beta (SEQ ID NO:3, 4) expression in CHO cells transfected with a mammalian expression vector into which the HGF-beta was cloned versus control medium.

[0048] FIG. 15 is a bar graph of pre-pro-B cell growth stimulating activity of the combination of rIL-7 (SEQ ID NO: 8) and rHGF-beta (SEQ ID NO:3, 4).

[0049] FIG. 16 is gel electrophoresis illustrating RT-PCT analysis of the HGF mRNA

(SEQ ID NO: l 1) transcripts from mouse BM stromal cells. First-stand cDNA from cultured IL-7 (-/-) mouse BM stromal cells subjected to PCR with primers designed to amplify the entire coding sequence of mouse HGF. The blot demonstrates both the 2230 bp product corresponding to the full-length HGF and a novel 840-bp product, corresponding to HGF-beta chain.

[0050] FIG. 17 is a western blot illustrating that recombinant IL-7 (SEQ ID NO: 8) forms a heterodimer with rHGF-β (SEQ ID NO:3, 4) in the presence of low molecular weight heparan- sulfate derived oligosaccharides. Equimolar concentrations of rIL-7 and rHGF-beta were mixed in the presence and absence of low molecular weight heparan sulfate-derived oligosaccharides. One hour later the mixtures were electrophoresed and developed with (A) anti-HFG-β Ab or (B) anti-IL-7 mAb. In each instance as shown, a 45 kD heterodimer was observed in the presence (lane 2), but not the absence (lane 1) of, heparan sulfate.

[0051] FIGS. 18-21 are flow cytometry histograms of B-cell populations (pre-pro-B,

(Fr.A), pro-B (Fr.B-C), and pre-B (Fr.C)) (FIG. 18) in IL-7 knock-out (K/O) mice with population fractions demonstrating expression of IL-7R-alpha (FIG. 19), TdT (FIG. 20), and Ομ (FIG. 21).

[0052] FIG. 22 are a series of flow cytometry histograms of B-cell populations demonstrating the effect of in vivo injection of rIL-7 into IL-7 KO mice on the development of B-lineage cells in BM. IL-7 K O mice were injected i.p. with 40 ng rIL-7 (SEQ ID NO:8) daily for 4, 7, 9, or 12 days. BM cells were harvested on day 0 or 1 day after the end of each series of injections, and were subjected to FCM analysis. (A) The B220+ population was subdivided into CD43+ (Fr. A-C) and CD43- (Fr. D-F) cells according to relative fluorescence intensity. (B) The B220+CD43+ population was subdivided into Fr. A, B-C and C according to relative

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1 fluorescence intensity for HSA. (C) The B220+CD43- population was separated into Fr. D, E and F according to relative fluorescence intensity for slgM. The relative numbers of cells in each of these fractions is expressed as the percentage of total nucleated BM cells per femur. (n.d.=not determined).

[0053] FIGS. 23 provides bar graphs demonstrating the ability of rIL-7 (SEQ ID NO: 8) to support the generation of B220+CD43+ cells from IL-7 K/O mice in vitro. 1 xlO⁶ BM cells from IL-7 K/O (-/-) mice, obtained (A) before or (B) 4 days after the onset of in vivo treatment with rIL-7, were incubated with medium (RPMI 1640 supplemented with 5% FBS and 5x10^"5 M 2 ME) containing the indicated concentrations of rIL-7. BM cells from untreated IL-7 competent (+/+) and RAG-1 K/O (-/-) mice were incubated under the same culture conditions. Cells were harvested 4 days later and the results were expressed as the ratio of the input and output numbers of B220+ CD43+ cells/well.

[0054] FIG. 24 is a bar graph demonstrating the ability of BM stromal cells from IL-

7(+/+) or IL-7(-/-) mice to support the in vitro generation of B-lineage cells from IL-7 K/O mice in the presence or absence of rIL-7. lxl 0⁶ BM cells from IL-7(+/+) and IL-7(-/-) mice were incubated in the presence or absence of 10 ng/ml rIL-7 (SEQ ID NO:8) on stromal cell layers established from either IL-7(+/+) or IL-7(-/-) mice. Cells were harvested on day 10 and the number of B220+ cells per well were calculated.

[0055] FIG. 25 provides flow cytometry histograms demonstrating phenotypic analysis of B-lineage cells generated in vitro on IL-7(+/+) BM stromal cells in the presence or absence of rIL-7 (SEQ ID NO:8). Day 10 culture-generated B-lineage cells from IL-7(+/+) and IL-7(-/-) mice (see FIG. 23, +/+ stromal cells) were analyzed for the expression of B220, CD43 and IL- 7R-alpha. (A) Proportions of CD43+ (Fr. A-C) and CD43- (Fr. D-F) cells among the B220+ population. (B) Proportions of IL-7R-alpha^/o and IL-7R-alpha*' cells among the B220+CD43+ population. Fewer than 10% Fr. D-F and 5% IL-7R-alpha^/!' cells were present in the input population (day 0);

[0056] FIG. 26 is a bar graph demonstrating the ability of culture-generated B-lineage cells from IL-7 K/O mice to respond to rIL-7 alone (SEQ ID NO: 8). lxl 0⁵ B-lineage cells from day 10 primary cultures containing +/+ stromal cells and 10 ng/ml rIL-7 (see FIG. 23) were transferred into secondary cultures containing medium only or medium plus 10 ng/ml rIL-7. Cells were harvested 4 days later and the numbers of B220+ CD42+ cells/well were determined. Results are expressed as the ratio of output to input numbers of cells/well.

[0057] FIG. 27 is a bar graph illustrating that CM plus rIL-7 (SEQ ID NO: 8) supports the proliferation of B220+CD43+ cells from IL-7 K/O mice in vitro. lxlO⁶ BM cells from IL-7 K/O mice were incubated for 20 days in medium (RPMI 1640 supplemented with 20% FBS) containing CM from WT BM stromal cells, rIL-7 (10 ng/ml), or both. The CM was either depleted by adsorption with anti-IL-7 mAb or sham-depleted with an isotype control antibody. The cultures were re-fed (50% volume) twice weekly.

[0058] FIG. 28 is a bar graph demonstrating that purified PPBSF plus rIL-7 (SEQ ID

NO:8) supports the proliferation of B220+CD43+ cells from IL-7 K/O mice in vitro. 1 x 10⁶ BM cells from IL-7 K/O mice were incubated for 20 days with medium containing purified PPBSF (10 ng/ml), rIL-7 (10 ng/ml), or both.

[0059] FIG. 29 is a bar graph illustrating the effect of pretreatment in vitro with purified

PPBSF, enabling B220+CD43+ cells from IL-7 K/O mice to proliferate in response to rIL-7 (SEQ ID NO:8) alone. lxlO⁶ BM cells from IL-7 K/O mice were incubated for 5 days with medium containing purified PPBSF (10 ng/ml), or rIL-7 (10 ng/ml). The cells from these primary cultures were transferred into secondary cultures and incubated for another 15 days in medium containing rIL-7 only (10 ng/ml).

[0060] FIG. 30 Cloning strategy for ligation of the IL-7 (SEQ ID NO: 10, 12), linker, and

HGF-beta coding sequences (See SEQ ID NO:9, 10) into baculovirus transfer vector pAcGP67A. The gp67 secretion sequence, IL-7, linker and HGF-beta DNA are constructed by overlapping PCR as described herein.

[0061] FIG. 31 Stimulation of mouse bone marrow (BM) cells by rIL-7 (SEQ ID NO:8) and/or scIL-7/HGF-beta in vitro. Freshly harvested BM cells from IL-7(-/-) mice were cultured in RPMI- 1640 containing 2-ME in the presence of 10 ng/ml rIL-7 or 30 ng/ml scIL-7/HGF-beta, or both. Nonadherent cells were harvested at day 17 and analyzed by flow immunocytometry. Top row shows representative histograms of B220+ and B220- cells in each culture. The vertical standards indicate the peaks (or theoretical peak; dashed line) of fluorescence intensity and are used to eliminate most of the overlap regions between the peaks. Middle row shows the contour plots for CD43 and HS A of the B220- and B220+ cells to the left and right of the peaks in the top row. The various fractions of developing B-lineage cells and their relative proportions in the B220- and B220+ cell subsets are indicated for each quadrant. Bottom row shows the relative proportion of fractions A₀ (CLPs), A_\ (early pre-pro-B cells) and A₂ (late pre-pro-B cells).

[0062] FIG. 32 Incorporation of BrdU by culture-generated BM lymphoid cells stimulated or cross-stimulated in vitro with rIL-7 (SEQ ID NO: 8) or scIL-7/HGF-beta. BM cells from IL-7(-/-) mice were cultured in the presence of rIL-7 (10 ng/ml) or scIL-7/HGF-beta (30 ng/ml) for 19 days. The cells were washed, cytokine-starved for 5 hours, stimulated with the homologous or heterologous cytokine for 3 hours, pulsed with BrdU, and stained with combinations of antibodies to B220, HAS, AA4.1, CD43, CD4, and BrdU. (A, C) Distribution early B-lineage subsets in each culture system. (B, D) Percentage of BrdU+ cells in each fraction of B-lineage cells. (A, B)■ indicates scIL-7/HGF-beta-generated cells stimulated with scIL-7/HGF-beta; m indicates IL-7-generated cells stimulated with IL-7. (C, D)■ indicates scIL-7/HGF-beta-generated cells stimulated with IL-7; m indicates IL-7-generated cells stimulated with scIL-7/HGF-beta. Means of duplicate samples are shown. Data are from 1 representative experiment of 2.

[0063] FIG. 33 Ability of antibodies to the IL-7 Recptor (R) and/or c-Met to inhibit the stimulation of mouse bone marrow cells by rIL-7 (SEQ ID NO:8) or scIL-7/HGF-beta. Culture- generated BM cells (4 x 10⁵ cells/well) from IL-7(-/-) mice were incubated for 3 days in the presence of rIL-7 (10 ng/ml) or scIL-7/HGF-beta (30 ng/ml) to which antibodies against IL-7R- alpha, yc, and/or c-Met (10 μg/ml) were added. Incorporation of [methyl-3H] thymidine (mean counts per minute [CPM] ± SD) was determined after a 12-hour pulse. * <.05 between antibody-treated and untreated values (similar results were obtained with isotype controls). **P<.05 versus value for anti-IL-7R-alpha or anti-HGFR alone. One representative experiment of 4 is shown.

[0064] FIG. 34 Analysis of the purified IL-7/HGF-p receptor proteins. The IL-7/HGF-p receptor complex was isolated on a scIL-7/HGF-beta affinity gel from purified culture-generated CLP/pre-pro-B cells. The eluates were subjected to SDS-PAGE under reducing (R) or nonreducing (NR) conditions, and Western blotting was done with antibodies to IL-7R-alpha, yc, or c-Met.

[0065] FIG. 35 Ability of rIL-7 (SEQ ID NO:8) or scIL-7/HGF-p to activate Jak3 and/or c-Met in mouse B-lineage bone marrow cells. B-lineage cells generated in cultures of IL-7(-/-) mouse BM cells supplemented with rIL-7 or scIL-7/HGF-P were harvested, placed in cytokine- free medium for 5 hours, and then stimulated with the homologous cytokine for 10 or 30 minutes. The supernatants form lysed cells were immunoprecipitated with anti-Jak3 or antiphosphotyrosine antibody and subjected to SDS-PAGE and Western blotting using the indicated antibodies. Arrows indicate phosphor-c-Met or phosphor- Jak3. Phosphorylation of Jak3 was used as an indicator of IL-7R signaling, and positive Western Blotting with anti-c-Met phosphospecific antibody as an indicator of c-Met signaling. The figures indicates that both rlL- 7 and scIL-7/HGF-beta signal through the IL-7R, but that only scIL-7/HGF-beta signals through c-Met as well.

[0066] FIG. 36 Signal transduction via a phosphotyrosine pathway. To demonstrate that scIL-7/HGF-p signals through a different pathway from IL-7 (SEQ ID NO:7, 8) or HGF alone (SEQ ID NO: 5, 6) tyronsine phosphorylation of whole cell lystates was analyzed by Western Blot developed with anti-phosphotyrosine antibodies. The results indicate differences in phosphorylation profiles between the IL-7-stimulated and HGF-stimulated cells, and also the IL- 7/HGF"P stimulated cells. Most striking was the appearance of 2 novel bands of phosphorylated proteins of approximately 30 kDa and 140 kDa in the scIL-7/HGF-P-stimulated cells and the absence of a 180 kDa band (see arrows). These results are consistent with following non- limiting hypothesis; that IL-7/HGF-P induces "cross-talk" between the IL-7 and c-Met receptors, which in turn activates new signaling pathways and induces new biological functions than those induced by IL-7 and HGF alone.

[0067] FIG. 37 Homologs of Human Hepatocyte Growth Factor (SEQ ID NO:5)

Identified by BLASTP. Queried sequence contained the full length human hepatocyte growth factor (HGF) polypeptide; i.e., contains both a and β chains (FL β = V495 to S728 (SEQ ID NO: 14); active peptide: V495-L511 (SEQ ID NO: 13) (NCBI Accession: BAA14348; 728 aa). A. indicates homologous domain structure between human HGF and homologous proteins; B. list of HGF homologs including accession number, annotated name/description, and homology score.

[0068] FIG. 38 Homologs of Human IL-7 (SEQ ID NO:7) Identified by BLASTP.

Queried sequence contained the full length human IL-7 polypeptide; (NCBI Accession: NP_000871 ; 177aa). A. indicates homologous domain structure between human IL-7 and homologous proteins; B. list of IL-7 homologs including accession number, annotated name/description, and homology score. [0069] FIG. 39 rIL-7/HGF- inhibits the growth of localized colon cancer and melanoma cell tumors. BALB/c mice were injected s.c. with 2x10⁵ CT-26 colon cancer cells followed by intratumoral injections with (A) rIL-7/HGF-P (2.5, 5, 15, or 30 μg) or PBS, or (B) equimolar doses of rIL-7/HGF- (15 μg), rIL-7 (5 μg) and/or rHGF-β (10 μg) or PBS, at 2-day intervals between days 2-24 after tumor inoculation. (C) C57BL/6 mice were injected s.c. with lxlO⁵ B16F10 melanoma cells, followed by intratumoral injections with rIL-7/HGF- (15 μg) or PBS at 2-day intervals between days 2-16 after tumor inoculation. Tumors were measured twice weekly. The mean tumor volume (mm³) + S.D. at the indicated time points are shown. The data are representative of 2 independent experiments with 4-6 mice per group.

[0070] FIG. 40 Infiltration of CD4⁺ and CD8⁺ T cells and activated CD1 l c⁺ DCs into rIL-7/HGF"P-treated tumors. BALB/c mice were injected s.c. with CT-26 colon cancer cells, and treated with equimolar doses of rIL-7/HGF- (15 μg), rIL-7 (5 μg) and/or rHGFp (10 μg), or PBS as in Figure IB. Thirty days after tumor inoculation, the mice were euthanized and the tumors were removed. Single-cell suspensions from the tumors were analyzed by flow immunocytometry for (A) CDl lc⁺ DCs and CD4⁺ and CD8⁺ T cells; and (B) the expression levels of CD80 and CD86 on the CDl lc⁺ DCs. Data represent (A) mean numbers + SD of positive cells per mg tumor tissue from 4 to 6 mice per group; and (B) relative mean + SD fluorescence intensity (MFI) of CD80 and CD86 on DCs in cytokine or PBS-treated tumors. * PO.05 compared with PBS-treated group; ** PO.05 as compared with the rIL-7 and/or rHGF- β-treated groups.

[0071] FIG. 41 Treatment of localized tumors with rIL-7/HGF- increases the numbers of CD4⁺ and CD8⁺ T cells and CDl lc⁺ DCs in the draining lymph nodes (DLNs). Single-cell suspensions of DLNs from the cytokine-treated tumors (see Figure 2) were analyzed for the numbers of CD1 lc⁺ DCs, and CD4⁺ and CD8⁺ T cells. Data represent mean numbers of positive cells + SD from 4 to 6 mice per group. *P<0.05 as compared with the PBS-treated group; ** P<0.05 as compared with the rIL-7 and/or rHGF-β -treated groups.

[0072] FIG. 42 Treatment of localized tumors with rIL-7/HGF- enhances tumor- specific T cell responses. BALB/c mice were injected s.c. with CT-26 colon cancer cells, and treated with equimolar doses of rIL-7/HGF- (15 μg), rIL-7 (5 μg) and/or rHGFp (10 μg), or PBS as in Figure IB. Thirty days after tumor inoculation, the (A) DLNs and (B) spleens were harvested, and cocultured with irradiated CT-26 colon cancer cells or 66.1 breast cancer cells. ELISPOT assays were then performed for INF-y⁺ cells. Data represent mean number of spots/lxlO⁵ T cells ± SD. *P<0.05 as compared with the PBS-treated group; ** P<0.05 as compared with the rIL-7 and/or rHGFp-treated groups.

[0073] FIG. 43 rIL-7/HGF-p inhibits the formation of pulmonary metastases by colon cancer and melanoma cells. (A) BALB/c mice were injected i.v. with 2x10⁵ CT-26 colon cancer cells, followed by i.v. injections with equimolar doses of rIL-7/HGF-P (15 μg), rIL-7 (5 μg) and/or rHGF-β (10 μg) or PBS at 2-day intervals between days 2-18. (B) C57BL/6 mice were injected i.v. with 2x10⁵ B16F10 cells, followed by injections with rIL-7/HGF-p (15 μg) or PBS at 2-day intervals between days 2-18. (A, B) The mice were euthanized on day 21 after tumor cell inoculation, and the total tumor nodules visible at the surface of the lungs were counted under a dissecting microscope. * P<0.05 as compared with PBS-treated group. ** PO.05 as compared with the rIL-7 and/or HGFp-treated groups. The data are representative of 2 independent experiments with 4-6 mice per group.

[0074] FIG. 44 The antitumor and antimetastatic activities of rIL-7/HGF-P in nude mice.

Nude mice were injected s.c. with (A) 2xl 0⁵ CT-26 colon cancer cells or (C) lxlO⁵ B16F10 melanoma cells followed by intratumoral injections with rIL-7/HGF- (15 μg) or PBS at 2-day intervals between (A) days 2-24 and (C) days 2- 16 after tumor cell inoculation. Data represent

■a

mean tumor volume (mm ) + S.D. is shown; 5 mice per group. Nude mice were injected i.v. with 2x10⁵ (B) CT-26 colon cancer cells or (D) B16F10 melanoma cells followed by the i.v. injection of rIL-7/HGF-p (15 μg) or PBS at 2-day intervals between days 2-18. Data represent mean + S.D. numbers of tumor nodules in the lungs on day 21 ; 5 mice per group. *P<0.05 as compared with the PBS-treated group.

[0075] FIG. 45 rIL-7/HGF- treatment inhibits local tumor growth, in vivo. (A) P388D leukemia cells (2X10⁵), (B) Line 66.1 breast cancer cells (3X10⁵) were injected s.c. into DBA/2, and BALB/c mice, respectively. Beginning on the day of tumor challenge, groups of mice were injected with rIL-7/HGF- (15 ug/injection), or control vehicle (PBS) at 2-day intervals. Tumor diameters were measured twice weekly. Data are reported as mean tumor diameter + SD from 5 mice each group. [0076] FIG. 46 rIL-7/HGF-p treatment inhibits local tumor growth of prostate cancer, in vivo. C57BL/6 (B6) mice were injected s.c. with 5 X 10⁶ syngeneic TRAMP-C1 prostate cancer cells. When the mice developed palpable tumors, groups of the mice were injected i.p. with rlL- 7/HGF (15 ug/injection), or control vehicle (PBS) at 2-day intervals. Tumor sizes were measured periodically. Data are reported as mean tumor diameter + SD from 5 mice each group.

DETAILED DESCRIPTION

[0077] The present invention relates to the discovery that certain stromal cell-derived cytokines form hybrid molecules that modulate tumor growth and metastases. Previously, it was demonstrated that a recombinant hybrid cytokine comprising the bioactive portions of interleukin (IL)-7 and the β-chain of hepatocyte growth factor (HGF-β) stimulates the growth of short-term hematopoietic stem cells, common lymphoid progenitors, and immature B- and T-lineage cells.

[0078] This application claims priority to U.S. Provisional Patent Application Serial No.

61/379,101 filed September 1, 2010, which is incorporated by reference in its entirety for all purposes. In addition, the disclosures of the following U.S. Patent Applications are incorporated by reference in their entirety for all purposes: U.S. Patent Application Serial No.: 11/601,059, filed November 17, 2006, which is a continuation-in-part of U.S. Patent Application Serial No.: 10/792,645, filed March 3, 2004, which is a divisional application of U.S. Patent Application No.: 09/823,933, filed March 30, 2001, now U.S. Patent No. 6,749,847; which claims priority under 35 U.S.C. § 1 19(e) to U.S. Provisional Patent Application No. 60/193,273, filed on Mar. 30, 2000.

[0079] Presently, it is demonstrated that the in vivo administration of IL-7/HGF- markedly inhibited both local tumor growth and formation of pulmonary metastases in animal cancer models, e.g., murine breast cancer, colon cancer, leukemia, melanoma, and prostate cancer. The antitumor effect of IL-7/HGF- correlated with a marked increase in the number of tumor-infiltrating CD4⁺ and CD8⁺ T cells and activated dendritic cells (DCs). A major role for these immune cells in tumor suppression was indicated by the inability of rIL-7/HGF-p to inhibit the growth of tumor cells in vitro and in congenitally athymic mice. In addition, analysis of interferon-γ (INF-y)-secreting T cells demonstrated that the immune response was tumor- specific. These findings demonstrate that the IL-7/HGF-P hybrid cytokine compositions provided by the invention are advantageous for use as a broad spectrum anti-tumor therapeutic. [0080] The following is a detailed description of the invention provided to aid those skilled in the art in practicing the present invention. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0081] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0082] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and described the methods and/or materials in connection with which the publications are cited.

[0083] It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural references unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning.

[0084] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references, the entire disclosures of which are incorporated herein by reference, provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2^nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5 ¹ Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, the Harper Collins Dictionary of Biology (1991). As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0085] The term "about" as it is used herein, in association with numeric values or ranges, reflects the fact that there is a certain level of variation that is recognized and tolerated in the art due to practical and/or theoretical limitations. For example, minor variation is tolerated due to inherent variances in the manner in which certain devices operate and/or measurements are taken. In accordance with the above, the phrase "about " is normally used to encompass values within the standard deviation or standard error.

[0086] As used herein, "derivatives" are compositions formed from the native compounds either directly, by modification, or by partial substitution. As used herein, "analogs" are compositions that have a structure similar to, but not identical to, the native compound.

[0087] The term "polypeptides" can mean, but is in no way limited to, recombinant full length, pro- and/or mature polypeptide forms as well as the biologically active forms, including fragments or splice variants, or recombinantly made truncations or portions derived from the full length polypeptides. Furthermore, polypeptides of the invention may include amino acid mimentics, and analogs. Recombinant forms of the chimeric polypeptides can be produced according to standard methods and protocols which are well known to those of skill in the art, including for example, expression of recombinant proteins in prokaryotic and/or eukaryotic cells followed by one or more isolation and purification steps, and/or chemically synthesizing cytokine polypeptides or portions thereof using a peptide sythesizer.

[0088] The term "effective amount/dose," "pharmaceutically effective amount/dose,"

"pharmaceutically effective amount/dose" or "therapeutically effective amount/dose" can mean, but is in no way limited to, that amount/dose of the active pharmaceutical ingredient sufficient to prevent, inhibit the occurrence, ameliorate, delay or treat (alleviate a symptom to some extent, preferably all) the symptoms of a condition, disorder or disease state. The effective amount depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 1000 mg/kg body weight/day of active ingredients is administered dependent upon potency of the agent. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0089] The term "pharmacological composition," "therapeutic composition,"

"therapeutic formulation" or "pharmaceutically acceptable formulation" can mean, but is in no way limited to, a composition or formulation that allows for the effective distribution of an agent provided by the invention, which is in a form suitable for administration to the physical location most suitable for their desired activity, e.g., systemic administration.

[0090] Non-limiting examples of agents suitable for formulation with the, e.g., nucleic acids provided by the instant invention include: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P- glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, for example the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol, 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Other non-limiting examples of delivery strategies, including CNS delivery of nucleic acid molecules include material described in Boado et al, 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al, 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al, 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058. All these references are hereby incorporated herein by reference.

[0091] The term "pharmaceutically acceptable" or "pharmacologically acceptable" can mean, but is in no way limited to, entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.

[0092] The term "pharmaceutically acceptable carrier" or "pharmacologically acceptable carrier" can mean, but is in no way limited to, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0093] The term "systemic administration" refers to a route of administration that is, e.g., enteral or parenteral, and results in the systemic districution of an agent leading to systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect. Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful.

[0094] The term "local administration" refers to a route of administration in which the agent is deliverd to a site that is apposite or proximal, e.g., within about 10 cm, to the site of the lesion or disease.

[0095] The term "nucleotide" can mean, but is no way limited to, a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, for example, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5- methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6- azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetyltidine, 5-(carboxyhydroxymethyl)uridine, 5'- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D- galactosylqueosine, 1 -methyladenosine, 1 -methylinosine, 2,2-dimethylguanosine, 3- methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7- methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5- methylcarbonyhnethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6- isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).

[0096] The term "nucleic acid" or "polynucleotide" can mean, but is in no way limited to, a molecule having more than one nucleotide, and is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules, analogs of DNA or RNA, including locked nucleic acids and peptide nucleic acids, and derivatives thereof.The nucleic acid can be single, double, or multiple stranded and can comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. The nucleic acids of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues in vitro, ex vivo, or in vivo through injection or infusion pump, with or without their incorporation in biopolymers.

[0097] A polynucleotide can be a DNA molecule, a cDNA molecule, genomic DNA molecule, or an RNA molecule. A polynucleotide as DNA or RNA can include a sequence wherein T (thymidine) can also be U (uracil). If a nucleotide at a certain position of a polynucleotide is capable of forming a Watson-Crick pairing with a nucleotide at the same position in an anti-parallel DNA or RNA strand, then the polynucleotide and the DNA or RNA molecule are complementary to each other at that position. The polynucleotide and the DNA or RNA molecule are substantially complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hybridize with each other in order to effect the desired process. [0098] The term "modified bases" can mean, but is in no way limited to, nucleotide bases other than adenine, guanine, cytosine and uracil at Γ position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule. The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-0- methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163).

[0099] The term "derivatives" can mean, but is in no way limited to, chemical compositions, for example, nucleic acids, nucleotides, polypeptides or amino acids, formed from the native compounds either directly, by modification, or by partial substitution. The term "analogs" can mean, but is in no way limited to, chemical compositions, for example, nucleic acids, nucleotides, polypeptides or amino acids that have a structure similar to, but not identical to, the native compound.

[00100] The term "hybridization" can mean, but is in no way limited to, the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under low, medium, or highly stringent conditions, including when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[00101] The term "conservative mutations" refers to the substitution, deletion or addition of nucleic acids that alter, add or delete a single amino acid or a small number of amino acids in a coding sequence where the nucleic acid alterations result in the substitution of a chemically similar amino acid. Amino acids that may serve as conservative substitutions for each other include the following: Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); hydrophilic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Hydrophobic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C). In addition, sequences that differ by conservative variations are generally homologous.

[00102] The term "down-regulate" can mean, but is in no way limited to, the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more proteins, or activity of one or more proteins is reduced below that observed in the absence of an agent provided by the invention. For example, the expression of a gene can be decreased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by high levels of gene expression.

[00103] The term "up-regulate" can mean, but is in no way limited to, the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more proteins, or activity of one or more proteins is greater than that observed in the absence of an agent provided by the invention. For example, the expression of a gene can be increased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.

[00104] By "modulate" is meant that the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more proteins, or activity of one or more proteins is up- regulated or down-regulated, such that the expression, level, or activity is greater than or less than that observed in the absence of an agent provided by the invention.

[00105] The term, "gene" can mean, but is in no way limited to, a nucleic acid that encodes RNA, for example, nucleic acid sequences including but not limited to a segment encoding a polypeptide.

[00106] The term "complementarity" can mean, but is in no way limited to, the ability of a nucleic acid to form hydrogen bond(s) with another RNA sequence by either traditional Watson- Crick, Hoogsteen base pairing or other non-traditional types.

[00107] The term "binding" can mean, but is in no way limited to, the physical or chemical interaction, direct or indirect, between two molecules (e.g., compounds, amino acids, nucleotides, polypeptides, or nucleic acids). Binding includes covalent, hydrogen bond, ionic, non-ionic, van der Waals, hydrophobic interactions, and the like.

[00108] The term "equivalent" or "homologous" can mean, but is in no way limited to, nucleic acids or proteins including those naturally occurring DNA, RNA or amino acid molecules have homology (partial or complete) to an IL-7 gene (e.g., SEQ ID NO: 10 or 12) or protein (SEQ ID NO:7 or 8) or a gene encoding HGF-β (SEQ ID NO:9 or 11) or protein (SEQ ID NO: 13 or 14), respectively, with similar function as IL-7 and HGF-β in various organisms, including human, rodent, primate, rabbit, pig, protozoans, fungi, plants, and other microorganisms and parasites. The equivalent RNA sequence also includes, in addition to the coding region, regions such as 5 '-untranslated region, 3 '-untranslated region, introns, intron-exon junction and the like. By "homology" is meant the nucleotide sequence of two or more nucleic acid molecules or two or more nucleic acid or amino acid sequences is partially or completely identical. In certain embodiments the homologous nucleic acid or amino acid sequence has 30%, 40%, 50%, 60%, 70%, 80%), 90%, or 95% sequence similarity or identity to an IL-7 gene or protein or a HGF-β gene or protein, respectively. In certain embodiments, the invention provides a nucleic acid having 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% similarity or identity to a nucleic acid encoding an polypeptide selected from SEQ ID NOs.:3-8, or 13-14 or a bioactive portion thereof. In additional embodiments, the invention provides a polypeptide having 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% similarity or identity to polypeptide selected from SEQ ID NOs.: 3-8, or 13-14 or a bioactive portion thereof.

[00109] "Homologs" can be naturally occurring, or created by artificial synthesis of one or more nucleic acids having related sequences, or by modification of one or more nucleic acid to produce related nucleic acids. Nucleic acids are homologous when they are derived, naturally or artificially, from a common ancestor sequence (e.g., orthologs or paralogs). If the homology between two nucleic acids is not expressly described, homology can be inferred by a nucleic acid comparison between two or more sequences. If the sequences demonstrate some degree of sequence similarity, for example, greater than about 30%> at the primary amino acid structure level, it is concluded that they share a common ancestor. For purposes of the present invention, genes are homologous if the nucleic acid sequences are sufficiently similar to allow recombination and/or hybridization under low stringency conditions. In addition, polypeptides are regarded as homologous if they can be subsitued into the hybrid cytokine described by the invention and provide activity comparable to the wild type proteins in the hybrid cytokine.

[00110] As used herein "hybridization," refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under low, medium, or highly stringent conditions, including when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[00111] The term "RNA" can mean, but is in no way limited to, a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" or "2'-OH" is meant a nucleotide with a hydroxyl group at the 2' position of a D-ribo-furanose moiety.

[00112] The term "vectors" can mean, but is in no way limited to, any nucleic acid-based technique used to deliver a desired nucleic acid, for example, bacterial plasmid, viral nucleic acid, HAC, BAC, and the like for cloning, amplification, and/or expression of a gene. [00113] The term "cell" can mean, but is in no way limited to, its usual biological sense, and does not refer to an entire multicellular organism. The cell can, for example, be in vivo, in vitro or ex vivo, e.g., in cell culture, or present in a multicellular organism, including, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).

[00114] The term "host cell" can mean, but is in no way limited to, a cell that might be used to carry a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. A host cell can contain genes that are not found within the native (non-recombinant) form of the cell, genes found in the native form of the cell where the genes are modified and re-introduced into the cell by artificial means, or a nucleic acid endogenous to the cell that has been artificially modified without removing the nucleic acid from the cell. A host cell may be eukaryotic or prokaryotic. General growth conditions necessary for the culture of bacteria can be found in texts such as BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, Vol. 1 , N. R. Krieg, ed., Williams and Wilkins, Baltimore/London (1984). A "host cell" can also be one in which the endogenous genes or promoters or both have been modified to produce one or more of the polypeptide components of the complex of the invention.

[00115] As used herein, the term "cytokine" is used generally to refer to a broad class of biologically active peptides or polypeptides including cytokines, lymphokines, chemokines, growth factors, interleukins, interferons, and the like. Cytokines are characterized by considerable "redundancy", in that many cytokines can share similar functions. In a comparable manner, cytokines are also pleiotropic (acting on many different cell types). As such, a given cell type may express receptors for more than one cytokine, and/or different tissues can express receptors for the same cytokine. The biological actions of cytokines can be characterized generally as either autocrine (the cytokine acts on the cell that secretes it); paracrine (the action is restricted to the immediate vicinity of a cytokine's secretion); endocrine (the cytokine diffuses to distant regions of the body (carried by blood or plasma) to affect different tissues); or exocrine (if the cytokine is secreted or stored in a duct for delivery to a specific site of action).

[00116] In addition, as used herein, "cytokine polypeptides," encompasses recombinant full length or pre-pro-polypeptide forms, pro-polypeptide forms, the mature or processed polypeptide forms, as well as the biologically active forms, including naturally occurring or recombinantly made truncations or portions derived from the full length polypeptides. Furthermore, polypeptides of the invention may include amino acid mimentics, and analogs. Recombinant forms of the chimeric polypeptides can be produced according to standard methods and protocols which are well known to those of skill in the art, including for example, expression of recombinant proteins in prokaryotic and/or eukaryotic cells followed by one or more isolation and purification steps, and/or chemically synthesizing cytokine polypeptides or portions thereof using a peptide sythesizer.

[00117] The term "IL-7/HGF-P," refers to, but is in no way limited to, a protein complex which comprises both an IL-7 (Interleukin-7) polypeptide or bioactive portion thereof, and an HGF-β (Hepatocyte Growth Factor-beta) polypeptide or bioactive portion thereof, and biologically-active variants thereof, covalently and/or non-covalently bound, directly or indirectly; as well as a chimeric or fusion protein which includes at least the bioactive portions of IL-7 and HGF-β joined in a single, contiguous polypeptide chain (i.e., chimeric and fusion IL- 7/HGF" hybrid cytokine polypeptides).

[00118] In any of the embodiments described herein the term "linker" relates to linkers of any kind, which are suitable for the binding of polypeptides. Examples of such linkers include but are not limited to a disulfide-bridge connecting amino acids from both polypeptides; heparin or heparan sulfate-derived oligosaccharides (glycosoaminoglycans) connecting both polypeptides; bifunctional or chemical cross-linkers; and a peptide or polypeptide linker. The unimolecular protein can also be a chimera or fusion polypeptide. For example, a polypeptide featuring the bioactive portions of IL-7 and HGF-β can be fused with each other, either directly or through an amino acid linker. In certain embodiments, the chimeric IL-7/HGF- hybrid cytokine polypeptides of the invention comprise an amino acid linker sequence of from 1 to about 100 amino acids. In other embodiments, the linker introduces flexibility into the chimeric polypeptide in order to relieve steric hindrance or to introduce a functional site (e.g., site of post- translational modification).

[00119] As used herein, "bioactive" or "biologically active" refers to the ability of a cytokine to effectuate a physiological change or response. The response may be detected, for example, at the cellular level, for example, as a change in gene expression, protein quantity, protein modification, protein activity, or combination thereof; at the tissue level; at the systemic level; or at the organism level. Techniques used to monitor these phenotypic changes include, for example, measuring: the binding of a ligand to its receptor in or on a cell, activation of cell signaling pathways, stimulation or activation of a cellular response, secretion or release of bioactive molecules from the cell, cellular proliferation and/or differentiation, or a combination thereof. Accordingly, the invention contemplates the construction and use of IL-7/HGF-p hybrid cytokines that comprise less than the full length amino acid sequence of the IL-7, and/or the HGF-β components of the hybrid cytokine. For example, in certain embodiments the hybrid cytokine comprises IL-7 with at least one domain from HGF-β selected from the group consisting of a Kringle domain, protease-like domain, and combinations thereof.

[00120] Using a long-term lymphoid BM culture system (LTBMC) (as described in Nakumra et al, Nature 342: 440-443 (1989), Rubin et al., Biophysica Acta 1 155: 357-371 (1993) and Zarnegar et al, J. Cell Biol. 129: 1177-1 180 (1995)) that selectively supports the proliferation, self-replication and differentiation of pre-pro-B cells from rat, mouse and human BM, monoclonal anitibodies were raised to a novel IL-7-associated growth factor that selectively stimulates proliferation of pre-pro-B cells and supports their differentiation to pro-B cells. As revealed by Western blot analysis, amino acid sequencing and molecular cloning, this naturally occurring pre-pro-B cell growth stimulating factor, is a covalently-bound heterodimer of IL-7 (SEQ ID NO:7, 8) and the beta-chain of hepatocyte growth factor (HGF) (SEQ ID NO:3, 4, 13, andl4).

[00121] The 30 kDa, non-IL-7 component of the PPBSF heterodimer (PPBSF-coF) is the beta-chain of the hepatocytic growth factor/scatter factor (HGF/SF). In addition, it is demonstrated that active artificially-derived PPBSF can be produced by expressing a chimeric polypeptide comprising HGF-β and IL-7, made by the cloning of the coding sequence of HGF-β with rIL-7 in a contiguous, single polynucleotide chain. This is a remarkable and wholly unexpected finding, inasmuch as HGF (of which the beta-chain is the mitogenic component) is one of the most important cytokines involved in the regulation of organogenesis in embryonic life and of tissue regeneration and repair in adult life.

[00122] Western blot analysis under reducing and nonreducing conditions directly demonstrates that PPBSF is a covalently-bound, 55 kDa, heterodimer. The heterodimer comprises a non-IL-7 co-factor (coF) of about 30 kDa (determined using monoclonal antibodies derived from PPBSF-immunized IL-7 KO mice). Western immunoblotting showed that PPBSF was a covalently-linked heterodimer of IL-7 (SEQ ID NO: 8) and a 30 kDa co-factor. Partial amino-terminal amino acid sequence analysis of purified PPBSF co-factor showed the first 15 of 17 amino acid residues were identical to the published sequence of HGF-beta chain (SEQ ID NO:3, 4). Western blot analysis confirmed the identity of PPBSF co-factor as the beta chain of HGF.

[00123] PPBSF-coF is constitutively produced by BM stromal cells from IL-7 K/O mice cultured under pro-B cell but not pre-B cell (i.e. Whitlock/Witte-type culture conditions), and "primes" pre-pro-B cells to proliferate in response to monomeric IL-7 in an anchorage- independent fashion by upregulating the expression of the IL-7 -a chain.

[00124] By both amino acid sequence analysis and reciprocal Western immunoblotting, it was discovered that the PPBSF-coF of PPBSF is the beta-chain of HGF (also described herein as "HGF-β"; SEQ ID NOs: 3, 4, 13). In confirmation of the same, the bioactivity of native PPBSF has been found to be neutralized by antibodies to the HGF-β chain (i.e., Val 496 to the end of the full lenth HGF protein; SEQ ID NO: 14). Although cDNA for pro-HGF had previously been cloned prior to the present invention, the beta-chain cDNA had not been isolated and cloned into appropriate expression vectors.

[00125] PCR amplification of the coding sequence of HGF (SEQ ID NO: 1 1) in stromal cells from IL-7 knock-out mice resulted in the application of two transcripts of 2230 and 840 bp. The smaller product showing complete homology with the published mouse HGF-β gene was subcloned into the mammalian expression vector pcDNA3.1 (+) and transfected into Chinese hamster ovary (CHO) cells. The HGF-beta gene was also subcloned into the prokaryotic fusion protein expression vector pCAL-n and transformed into E. coli BL21 (DE3).

[00126] The rHGF-β DNA was purified by calmodulin affinity resin. Unexpectedly, rIL-7 (SEQ ID NO: 8) spontaneously complexed with rHGF-β (SEQ ID NOs:3, and 4) in the presence of low molecular weight heparin sulfate (HS)-derived oligosaccharides (below about 3000 Dalton) to form a heterodimer having the functional activity of native PPBSF. However, because several naturally occurring variants of HGF-β produced by alternative splicing of the HGF gene have been identified, the precise form of HGF-β represented in PPBSF may vary. Therefore, in certain embodiments the chimeric cytokine provided by the invention comprises a polypeptide comprising 70%, 75%, 80%, 85%, or 95% in sequence identity to a polypeptide having a amino acid sequence of at least one of SEQ ID NOs: 3, 4, 13, or 14.

[00127] PPBSF has been found to selectively stimulate the proliferation of pre-pro-B cells and to support the generation of pro-B cells (the next recognized stage in early B-lymphocyte development). PPBSF "primes" pre-pro-B cells to proliferate in response to monomeric IL-7 (SEQ ID NO:7, 8) in an anchorage-independent fashion by upregulating the expression of the IL- 7R-a chain. PPBSF also upregulates the expression of terminal deoxynucleotidyl transferase (TdT) and initiates the expression of cytoplasmic immunoglobulin mi heavy chain (ομ). PPBSF also stimulates the proliferation of thymocytes.

[00128] We have tested the in vivo effect of local and systemic administration of rlL- 7/HGFp on the growth and metastasis of murine CT-26 colon cancer and B16F10 melanoma cells. The results demonstrated that the intratumoral injection of rIL-7/HGF-P significantly inhibited local tumor growth, apparently by inducing the expansion and activation of tumor- infiltrating lymphocytes (TILs) and DCs. In addition, the systemic administration of rlL- 7/HGF significantly inhibited the formation of tumor metastases in the lung.

[00129] The IL-7/HGF-P complex can be isolated from natural sources, e.g., mammalian tissues or cell lines which are known to be a source of cytokines or growth factors. It may also be formed from recombinant and/or natural components as shown herein. PPBSF was shown to be expressed by bone marrow stromal cells in our pro-B cell culture system. Alternatively, PPBSF can be reconstituted from products of prokaryotic or eukaryotic expression of exogenous DNA sequences i.e., derived by recombinant means.

[00130] The present invention also provides biologically-active variants, truncations, and fusion proteins of the IL-7 and/or HGF-β components of the hybrid cytokine. Such variants include any homologs, orthologs or paralogs to either IL-7 (SEQ ID NO:7, 8) or HGF-beta (SEQ ID NO:3, 4, 13, and 14; See FIGs. 37 and 38), including, for example, substitution analogs wherein one or more amino acids have been substituted with a different amino acid(s), deletion analogs wherein one or more amino acids have been deleted, and addition analogs wherein one or more amino acids have been added. Deletions and additions of one or more amino acids are made either within an internal region of the polypeptide or at the amino or carboxyl terminal ends. Additional potential variations provided include other heterodimeric (or multimeric) cytokine complexes containing IL-7 and/or HGF (alpha and/or beta chains), and other hybrid cytokines unrelated to either IL-7 or HGF, whether naturally occurring or artificially created, including those that bind to the receptors for HGF, IL-7, and/or yc.

[00131] Therefore, it is demonstrated that PPBSF is a covalently-linked heterodimer of IL- 7 and a 30 kDa cofactor by Western immunoblot analysis under reducing and non-reducing conditions; that PPBSF is a self-aggregating complex of IL-7 and a 30 kDa cofactor by addition of IL-7 to conditioned medium from IL-7 gene-deleted mice; that PPBSF, but not IL-7 or PPBSF cofactor alone, upregulates the expression of IL-7R-a, TdT and ομ on/in pro-B cells from IL-7 gene-deleted mice and "primes" then to proliferate in response to monomeric IL-7; and that the PPBSF cofactor is the beta chain of HGF/SF. Therefore, the description provides cloned, recombinant forms of the hybrid cytokine and demonstrates heterologous expression (prokaryotic and eukaryotic) of the same, as well as neutralizing monoclonal antibodies specific for the PPBSF cofactor; all of which are encompassed by the invention.

[00132] In addition, it is demonstrated that rIL-7 spontaneously complexes with rHGF- beta in the presence of low molecular weight heparin sulfate (HS)-derived oligosaccharides to form a heterodimer having the functional activity of native PPBSF; and that a chimeric IL- 7/HGF-P nucleic acid fusion construct, expressed as a single-chain protein, demonstrates efficacy and stability.

[00133] As described previously, complexes of recombinant IL-7 (SEQ ID NO:7, 8) and HGF-β (SEQ ID NO:3, 4, 13, and 14) formed in the presence of heparan sulfate-derived oligosaccharides are less stable then native IL-7/HGF- complexes. Without being limited to any particular theory, the inventors believe that the instability observed with the recombinant IL- 7/HGF-P complex is due to incomplete glycosylation. The instability of the recombinant IL- 7/HGF-P complex also makes it difficult to purify sufficient IL-7/HGF-p for large-scale in vitro or in vivo use. The instability of the complexed hybrid cytokine was overcome by constructing a chimeric IL-7/HGF-P polypeptide. The efficacy of the chimeric cytokine constructs have also been demonstrated. See, e.g., Lai et al. A recombinant single-chain IL-7/HGF-beta hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-S_]2, CLPs, and pre-pro-B cells. Blood. 2006; 107: 1776-1784, which is incorporated herein by reference in its entirety for all purposes.

[00134] Unlike recombinant IL-7 (SEQ ID NO:7, 8), which stimulated pro-B cells and pre-B cells only, the chimeric or single-chain (sc) IL-7/HGF-beta stimulated the proliferation of pre-pro-B cells, common lymphoid progenitors (CLPs), and colony-forming unit (CFU)-Si₂ in cultures of IL-7(-/-) mouse bone marrow (BM) cells. When injected in vivo, 3- to 4-fold more splenic B-lineage cells appeared in recipients of BM cells from the scIL-7/HGF-p-stimulated cultures than from rIL-7-stimulated cultures. Moreover, on a per-cell basis, scIL-7/HGF-P culture-generated cells produced 16- to 20-fold more BM and splenic B-lineage cells than did normal BM cells. Antibody blocking, receptor phosphorylation, and confocal microscopy demonstrated that scIL-7/HGF-P signals though both the IL-7 and HGF (c-Met) receptors, which form IL-7R/c-Met complexes on the surface of CLPs and pre-pro-B cells. In addition, the IL-7R- alpha chain, yC chain, and c-Met were coisolated from purified CLPs and pre-pro-B cells on scIL-7/HGF-P affinity gels, indicating that they are major components of the IL-7/HGF-P receptor. Hence, the present results demonstrate that the IL-7/HGF-P chimeric cytokine efficiently and selectively stimulates the most primitive B-lineage precursors in BM by inducing juxtacrine interactions between the IL-7 and c-Met receptors.

[00135] Much of what is known about the microenvironmental regulation of early B cell development stems from the use of long-term in vitro bone marrow culture systems. As discussed above, the present inventors have previously described a long-term lymphoid cell culture system that selectively generates large numbers of pre-pro-B cells and pro-B cells from rat, mouse, and human BM in the presence of mouse BM adherent cells (Nakamura et al., Nature 342: 440-443, 1989; Rubin J S et al. Biophysica Acta 1 155: 357-371 , 1993; Zarnegar R et al. 1995; J Cell Biol 129: 1 177-1 180; and Liu et al. Biophysica Acta 1216: 299-303; incorporated herein by reference) referenced herein as the pro-B cell culture. Approximately 50% of the lymphoid cells show partial D-J heavy chain Ig gene rearrangements; whereas the remaining lymphoid cells have a germline configuration, and are themselves derived from even more primitive, B220^", precursors. Unlike more traditional LTBMC systems, the pro-B cell culture system, so described, selectively generates pre-pro-B cells and early pro-B cells from adult BM, even when the lymphoid progenitors are separated from the adherent cell layer by a microporous membrane culture insert or cultured in stromal cell conditioned medium (CM). However, under optimal conditions, the "early" (TdT) pre-pro-B cells, adhere tightly to BM stromal cells, and self-replication occurs indefinitely upon serial transfer to new adherent cell layers in vitro. In addition, these pre-pro-B cells produce non-adherent pro-B cells in vitro and rapidly generate sIgM⁺ B cells after in vivo transfer. Yet, neither ομ⁺ pre-B cells nor sIgM⁺ B cells are produced in significant numbers in vitro, even in the presence of 2-ME.

[00136] Although there is no evidence of progressive clonal restriction or leukemic transformation under normal circumstances, the pro-B cell culture system is also able to generate leukemic pre-pro-B cells and pro-B cells in vitro, when seeded with BM cells from rats that have been infected neonatally with the Gross leukemia virus or from human patients with acute lymphoblastic leukemia (ALL). Like their normal counterparts, these B-cell lineage leukemias are dependent upon the presence of a BM adherent cell layer (or conditioned medium therefrom). Moreover, the cells that undergo leukemic transformation co-isolate on the FACS with the precursors that generate normal pro-B cells. They therefore have proved useful as target cells in bioassays for stage specific lymphoid growth-stimulating factors.

[00137] The sequential expression of low and high concentrations of high affinity IL-7R during early B-lineage development is analogous to events observed during early thymocyte development. Given that pro-B cells from yc gene-deleted mice express only low levels of IL-7R -alpha-chain, IL-7R-alpha KO mice apparently fail to generate pro-B cells, and excess IL-7 fails to increase pre-pro-B cell generation in vivo, it is possible that signal transduction through high affinity IL-7R (alpha/yc) is required to transmit a proliferative signal for pre-pro-B as well as pro-B cells. This is further supported by the inventors' recent observation that, although the IL- 7R-alpha is not upregulated on pro-B cells from IL-7(-/-) mice, its expression can be induced on such cells in vitro by purified PPBSF, but not rIL-7. Hence, PPBSF may favor the association of IL-7R-alpha and yc chains under conditions of low IL-7R-alpha expression, whereas monomeric IL-7 may require high concentrations of IL-7R-alpha.

[00138] There appears to be a "priming" effect of PPBSF for monomeric IL-7 (SEQ ID NO:7, 8), wherein PPBSF selectively regulates the Gj/S transition of pre-pro-B cells, and monomeric IL-7 selectively regulates the Gi/S transition of pro-B cells. Such sequential actions of PPBSF and monomeric IL-7 would correlate nicely with the demonstration of separate microanatomical niches, differential adhesion mechanisms, decreasing need for cognitive interactions, and increasing dependency on IL-7 during early B cell development. Compatible conclusions have been reached by Billips et al (Blood 79: 1185 (1992)) using the S17 stomal cell line; and Hayashi et al. (J. Exp. Med. 171 : 1683 (1990)), using the PA6 stromal cell line. Even more intriguing is the possibility that PPBSF may be involved in regulating the commitment of HSC to development along the B (and possibly T) lymphoid pathways. Hence, differences in molecular form and, possibly, site of expression may render IL-7 pre-pro-B cell stage-specific; and differences in the receptor-binding domain (IL-7 for HGF-alpha) may render HGF-beta (SEQ ID NO:3, 4, 13, and 14) lymphoid lineage-specific. [00139] Despite the occurrence of early B-lineage development in IL-7 K/O and IL-7R K/O mice, the present inventors postulate that IL-7 (SEQ ID NO:7, 8), in the form of PPBSF, is the preferred ligand under physiological conditions. Furthermore, the present inventors suspect that those compensatory mechanisms that do exist may be suboptimal, given that pro-B cells in IL-7(-/-) mice do not upregulate TdT or IL-7R-alpha during IgH gene rearrangement, do not initiate μ expression, and do not proliferate in response to monomeric IL-7. However, once stimulated with PPBSF, TdT and IL-7R-alpha are upregulated, cell proliferation is stimulated by monomeric IL-7, and ϋμ is expressed, at least in vitro. These results may help to explain why the in vivo administration of anti-IL-7 mAb prevents the development of pro-B cells in normal mice, namely by causing the coordinate elimination of IL-7 and PPBSF.

[00140] In addition to the above, currently demonstrated is that the local injection of recombinant hybrid IL-7/HGF-p cytokine at the site of tumor cell inoculation significantly inhibited tumor growth, and that the systemic administration of the IL-7/HGF-P hybrid cytokine inhibited the formation of pulmonary metastases in animal models of cancer, e.g., murine models of colon cancer and malignant melanoma. As would be understood by those of skill in the art, such model systems correlate well with in vivo activity in humans.

[00141] The antitumor effects of IL-7/HGF- were associated with increased percentages and numbers of TILs and activated DCs in the tumors. Furthermore, IL-7/HGF- enhanced tumor-specific regional and systemic T cell responses in lymph node (LN) and spleen, as demonstrated by differential stimulation of INF-y-secreting cells in vitro. IL-7/HGF-P failed to inhibit the growth of tumor cells in vitro. Without being bound by any particular theory, the data appears to suggests that IL-7/HGF-P inhibits local tumor cell growth indirectly, presumably by activating (and possibly attracting) T cells and DCs. In contrast, systemically administered IL- 7/HGF-P continued to inhibit the formation of metastases in nude mice, indicating that it also had a T cell-independent mechanism of action. For example, it is possible that IL-7/HGF- also stimulates other immune cells, such as NK and macrophages (Walser TC, Ma X, Kundu N, et al. Immune-mediated modulation of breast cancer growth and metastasis by the chemokine Mig (CXCL9) in a murine model. J Immunother 2007;30:490-498; Ostrand-Rosenberg S. Immune surveillance: a balance between protumor and antitumor immunity. Curr Opin Genet Dev 2008; 18: 11-8; Chan CJ, Andrews DM, and McLaughlin NM, et al. DNAM-1/CD155 interactions promote cytokine and NK cell-mediated suppression of poorly immunogenic melanoma metastases. J Immunol 2010, 184:902-1 1), which, in euthymic mice, work in concert with T cells to inhibit tumor cell growth and survival, especially in metastatic disease.

[00142] Several studies have suggested that IL-7 has antitumor activity (Hock H, Dorsch M, Diamantstein T, Blankenstein T. Interleukin 7 induces CD4 T cell-dependent tumor rejection. J Exp Med 1991 ;74: 1291-8; Aoki T, Tashiro K, Miyatake S, et al. Expression of murine interleukin 7 in a murine glioma cell line results in reduced tumorigenicity in vivo. Proc Natl Acad Sci U S A 1992;89:3850-4; Murphy WJ, Back TC, Conlon KC, et al. Antitumor effects of interleukin-7 and adoptive immunotherapy on human colon carcinoma xenografts. J Clin Invest 1993;92: 1918-24; Andersson A, Yang SC, Huang M, et al. IL-7 promotes CXCR3 ligand-dependent T cell antitumor reactivity in lung cancer. J Immunol 2009;182:6951-8; McBride WH, Thacker JD, Comora S, et al. Genetic modification of a murine fibrosarcoma to produce interleukin 7 stimulates host cell infiltration and tumor immunity. Cancer Res 1992; 52:3931-7; Li B, VanRoey MJ, Jooss K. Recombinant IL-7 enhances the potency of GM-CSF- secreting tumor cell immunotherapy. Clin Immunol 2007;123: 155-65; Pellegrini M, Calzascia T, Elford AR, et al. Adjuvant IL-7 antagonizes multiple cellular and molecular inhibitory networks to enhance immunotherapies. Nat Med 2009;15:528-36). We demonstrate that IL-7 could inhibit both tumor growth and the formation of metastases by CT-26 colon cancer cells. However, when given in the same molar amounts, our data show that IL-7/HGF-P was significantly more effective than IL-7 (and/or HGF-β) in inhibiting tumor formation. This difference may be qualitative as well as quantitative, as IL-7/HGF-P was able to activate tumor-infiltrating DCs, whereas IL-7 was not.

[00143] DCs express the receptors for both IL-7 and HGF (Fry TJ, Mackall CL. Interleukin-7: from bench to clinic. Blood 2002; 99:3892-904; Sonmez M, Ovali E, Dilonen T, et al. The role of hepatocyte growth factor in the differentiation of dendritic cells from peripheral blood monocytes. Saudi Med J 2007;8:688-95; Rutella S, Bonanno G, Procoli A, et al. Hepatocyte growth factor favors monocyte differentiation into regulatory interleukin (IL)- 10++IL-121ow/neg accessory cells with dendritic-cell features. Blood 2006;108:218-27; Okunishi K, Dohi M, Nakagome , et al. A novel role of hepatocyte growth factor as an immune regulator through suppressing dendritic cell function. J Immunol 2005;175:4745-53), and, as we have previously shown in dual receptor B- and T-lineage cells (Lai L., Zeff, RA, and Goldschneider I. A recombinant single-chain IL-7/HGF-p hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-Si₂, CLPs, and pre-pro-B cells. Blood 2006;107: 1776-84), juxtacrine interactions secondary to receptor cross-linking by IL-7/HGF-p may result in novel functional readouts, such as cell survival, activation, and maturation. Of course, it is also possible that IL-7/HGF-P may indirectly affect DCs by stimulating other cell types to generate DC-stimulatory factors (Zou GM, Tarn YK. Cytokines in the generation and maturation of dendritic cells: recent advances. Eur Cytokine Netw 2002;13: 186-9). In contrast, although both IL-7 and HGF have been shown to individually affect DCs, they primarily influence the development of immature DCs from intrathymic precursors and peripheral blood monocytes (Fry TJ, Mackall CL. Interleukin-7: from bench to clinic. Blood 2002; 99:3892-904; Varas A, Vicente A, Sacedon R, Zapata AG. Interleukin-7 influences the development of thymic dendritic cells. Blood 1998;92:93-10; Saunders D, Lucas K, Ismaili J, et al. Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor. J Exp Med 1996;184:2185-96; Marquez C, Trigueros C, Fernandez E, Toribio ML. The development of T and non-T cell lineages from CD34 human thymic precursors can be traced by the differential expression of CD44. J Exp Med 1995;181 :475-83; Li L, Masucci MG, Levitsky V. Effect of interleukin-7 on the in vitro development and maturation of monocyte derived human dendritic cells. Scand J Immunol 2000;51 :361-715, 19-22). In addition, IL-7 has been shown to down-regulate the expression of MHC II on DCs and to diminish the homeostatic proliferation of CD4⁺ T cells in a lymphopenic setting (Guimond M, Veenstra RG, Grindler DJ, et al. Interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of CD4+ T cells. Nat Immunol 2009;10: 149-57). HGF also can affect DC development, although the results vary. On the one hand, HGF has been reported to induce CD34⁺ BM cells to generate DCs that could stimulate T cell proliferation (Sonmez M, Ovali E, Dikmen T, et al. The role of hepatocyte growth factor in the differentiation of dendritic cells from peripheral blood monocytes. Saudi Med J 2007;8:688-95).

[00144] On the other hand, HGF has been found to favor the development of tolerogenic DCs from monocytes (Rutella S, Bonanno G, Procoli A, et al. Hepatocyte growth factor favors monocyte differentiation into regulatory interleukin (IL)-10++IL-121ow/neg accessory cells with dendritic-cell features. Blood 2006;108:218-27) and to suppress DC functions such as antigen- presentation (Okunishi K, Dohi M, Nakagome K, et al. A novel role of hepatocyte growth factor as an immune regulator through suppressing dendritic cell function. J Immunol 2005;175:4745- 53). Similarly, although many studies have shown that IL-7 can directly stimulate the survival and proliferation of T cells (Fry TJ, Mackall CL. Interleukin-7: from bench to clinic. Blood 2002; 99:3892-904), our data showed that rIL-7/HGF- treatment induced the appearance of significantly larger numbers of CD4⁺ and CD8⁺ T cells in the tumors and DLNs than did rIL-7 treatment (with or without rHGF-β). This may be related to the activation of DCs by rlL- 7/HGFp, but not rIL-7. Hence, rIL-7/HGF-P not only has distinct advantages over its component cytokines regarding the activation of DCs, but, as a likely consequence, the activation of antigen- specific T cells as well.

[00145] Importantly, many studies have shown that HGF/c-Met signaling plays a major role in stimulating the growth, invasion and metastasis of a wide variety of cancers in vivo, and that it also stimulates the proliferation of tumor cells in vitro (Birchmeier, C. et al. Met, metastasis, motility and more. Nat Rev Mol Cell Biol 2003; 4: 915-25). This of course raised concerns that HGFp, alone or when complexed with IL-7, might do the same. However, the present study with colon cancer and melanoma, show that this is not the case. Rather, our results show that HGF-β, whether given alone or mixed with rIL-7, did not stimulate tumor cell growth in vitro, even though the cells expressed c-Met. This is consistent with reports that HGF-β binds to c-Met and is required for the mitogenic activity of HGF, but it does not by itself stimulate cell growth (Kirchhofer D, Yao X, Peek M, et al. Structural and functional basis of the serine protease-like hepatocyte growth factor beta-chain in Met binding and signaling. J Biol Chem 2004;279:39915-2; Lokker NA, Mark MR, Luis EA, et al. Structure-function analysis of hepatocyte growth factor: identification of variants that lack mitogenic activity yet retain high affinity receptor binding. EMBO J 1992;1 1 :2503-10). Even more reassuring, our results show that the HGFp component of IL-7/HGF-P appears to enhance the anti-tumor effects of the IL-7 component. Therefore, whether used alone or in combination with other therapeutic agents, IL-7/HGF"P not only may prove useful in the treatment of cancer, but does not appear to pose a risk of provoking tumor growth (or recurrence) if used in the treatment of non-neoplastic lymphohematopoietic disorders.

[00146] As would be understood by one of ordinary skill in the art, a hybrid cytokine of as described herein could be used alone or in conjunction with other factors to treat a number of hematopoietic disorders, in vivo, in a subject, e.g., human beings and/or domesticated animals, that result from disease or injury to B-lineage (and other) cells in bone marrow. These include the following: pancytopenia, myelodysplastic syndrome, leukemias and lymphomas, hereditary or acquired immunodeficiency disorders, and myelosuppression resulting from radiation treatment, chemotherapy, drug allergies, or environmental toxins. A hybrid cytokine of the invention will also be useful in expanding and/or enhancing engraftment of B-lineage progenitor cells in vivo syngeneic, allogeneic or autologous bone marrow transplantation, or ex vivo in marrow or HSC cultures prior to transplantation. Further, such treatment will reduce the period of depressed immunity due to delayed B cell regeneration that frequently is experienced by patients after transplantation. Also, a hybrid cytokine as provided herein will enhance the engraftment of genomically modified B-cell precusors in the treatment of selected immunodeficiency and leukemic disorders. In addition, a hybrid cytokine as described herein can be used to enhance the growth of leukemic B-lineage cells in vitro to permit customized screening profiles of chemotherapeutic and immunotherapeutic sensitivity to be developed for individual patients, or to permit individualized tumor vaccines to be produced. Furthermore, a deficiency or abnormality of PPBSF itself may prove to be a cause of immunodeficiency in some patients, making screening assays for PPBSF useful.

[00147] In certain embodiments, a hybrid cytokine of the invention is used in a method for bone marrow transplantation comprising pretreating the marrow prior to transplantation and/or may be administered in vivo after transplantation. The hybrid cytokine of the invention may be used as a pharmacological agent itself or introduced by way of a transformed cell, viral vector, etc. The hybrid cytokine of the invention proffers significant therapeutic advantages to the bone marrow recipients in that it substantially increases lymphocyte precursors. Bone marrow recipients usually take months to approach normal levels of B and T-lymphocytes after transplantation. The hybrid cytokine of the invention has been seen not only to stimulate parental cells to generate large numbers of mature progeny, but to produce more parental cells (self- replication), leading to long-term engraftment.

[00148] Animal studies suggest that the hybrid cytokine as provided herein will be useful in the treatment of acute lymphoblastic leukemia in that it has been found to proliferate leukemic as well as normal cells. In certain embodiments, the invention provides a method for treating leukemia comprising administering a composition comprising an effective amount of a hybrid cytokine as described herein to leukemic patients, wherein the malignant cells are activated to proliferate and/or differentiate. As most chemotherapeutic agents today are designed to selectively kill dividing cells, such chemotherapeutic agents in conjunction with a hybrid cytokine of the invention provide a better "kill rate" of the malignant cells (a certain portion of the population of malignant cells usually are non- dividing at time of chemotherapy and therefore are protected from the cytotoxic effect of the chemotherapeutic agents).

[00149] Because the hybrid cytokine as described herein also stimulates proliferation of immature thymocytes, in vivo, it can be equally useful in treating disorders of T lymphocytes as well as B lymphocytes. Indeed, in certain embodiments, the description provides methods for treating T lymphocytic diseases comprising administering a composition comprising an effective amount of the hybrid cytokine as described herein, wherein the hybrid cytokine is effective for inducing commitment of HSCs to bipotential lymphoid differentiation, and wherein, the hybrid cytokine is effective to treat and/or correct severe combined immunodeficiency disorders, e.g., AIDS.

[00150] In addition, the description provides methods for inhibiting cancer cell growth and/or metastasis, in vivo, comprising administering a composition comprising an effective amount of a hybrid cytokine as described herein, and a pharmaceutically acceptable carrier, wherein the cancer cell is not a B cell lymphocytic cancer cell. In certain embodiments, the hybrid cytokine comprises a non-covalently or covalently bound complex of IL-7 and HGF-β or bioactive portions thereof. In additional aspects, the description provides methods of treating or inhibiting the growth and/or metastasis of a cancer cell wherein the cancer cell is at least one of a colon cancer cell, melanoma cancer cell, or T cell lymphocytic cell. In another embodiment, the hybrid cytokine comprises a complex of 11-7 and at least one bioactive portion of HGF-β selected from the group consisting of a Kringle domain, protease-like domain, and combinations thereof.

[00151] In another embodiment, the hybrid cytokine is a chimeric IL-7/HGF-P hybrid cytokine or fusion protein comprising an IL-7 polypeptide or bioactive portion thereof, and an HGF-β polypeptide or bioactive portion thereof, wherein the individual polypeptide components or portions are joined contiguously in a single polypeptide chain. In still other embodiments, the chimeric hybrid cytokine polypeptide or fusion protein further comprises an amino acid linlcer disposed between the IL-7 polypeptide and the HGF-β polypeptide. In certain embodiments, the linker is from 1 to about 100 amino acids in length. [00152] In certain embodiments of the methods of inhibiting cancer cell growth and/or metastasis, the therapeutic composition comprising an effective amount of the hybrid cytokine as described herein, is administered to a localized tumor cancer cell, e.g., via local administration. In still other embodiments, the cancer cell is a metastatic cancer cell. In addition, the methods described herein contemplate any suitable route of administration. In certain embodiments, where a localized tumor is to be treated, the therapeutic composition as described herein is administered directly to or near the site of the tumor, e.g., subcutaneously. Still in other preferred embodiments, where a metastatic cancer is to be treated, the therapeutic composition as described herein is administered systemically, e.g., intravenously.

[00153] In a further aspect, the description provides methods of increasing the number of tumor-infiltrating lymphocytes (TILs) and/or Dendritic Cells (DCs), in vivo, comprising administering a composition comprising an effective amount of the hybrid cytokine as described herein, to an individual suffering from cancer. In an embodiment, the TILs comprise CD4+ and/or CD8+ T cell lymphocytes. In an additional embodiment, the DCs comprise CD80+ and/or CD86+ DCs.

[00154] In another aspect, the description provides methods of creating a tumor-specific T cell, in vivo, comprising administering a composition comprising an effective amount of the hybrid cytokine as described herein, to an individual suffering from cancer, wherein the hybrid cytokine is effective for enhancing the secretion of interferon-gamma (IFN-γ) by the T cell.

[00155] In still another aspect, the invention provides methods of inhibiting pulmonary metastatic disease, in vivo, comprising administering a composition comprising an effective amount of the hybrid cytokine as described herein, and a pharmaceutically acceptable carrier to an individual suffering from pulmonary metastatic disease, wherein the hybrid cytokine is effective for inhibiting the growth and/or proliferation of pulmonary metastases.

[00156] In any of the methods descrbied herein, the hybrid cytokine is a chimeric or fusion protein comprising an IL-7 polypeptide, homolog, or bioactive portion thereof, and an HGF-β polypeptide, homolog, or bioactive portion thereof, joined contiguously in a single polypeptide chain.

[00157] In still another aspect, the description provides therapeutic compositions comprising an effective amount of a hybrid cytokine as described herein, in combination with an effective amount of at least one other biologically active agent, such as, for example, an anti- oncogenic agent, anti-angiogenic agent, or the like. Accordingly, the description also provides methods for inhibiting cancer cell growth and/or metastasis, in vivo, comprising administering a composition comprising an effective amount of a hybrid cytokine as described herein, together with at least one other biolocially active agent, and a pharmaceutically acceptable carrier, wherein the composition is effective in inhibiting the growth, proliferation, and/or metastasis of a cancer cell.

[00158] Specific examples of biologically beneficial ingredients that can be utilized in any of the embodiments described herein include: hyaluronic acid, growth factors (e.g. VEGF, TGF family), therapeutic antibodies (e.g., Humira), substance P, glucosamine, chondroitin sulphate, glycosaminoglycans, pain control agents (e.g morphine), synovial fluid and/or its components, steroids and derivatives. It should further be understood that the combinations which are to be included within this invention are those combinations useful for their intended purpose. The agents set forth below are illustrative for purposes and not intended to be limited. The combinations which are part of this invention can be the compositions provided by the present invention and at least one additional agent selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional agents if the combination is such that the formed composition can perform its intended function.

[00159] Thus, in additional embodiments, the compositions provided by the invention can optionally further comprise an effective amount of at least one compound or protein selected from at least one of an anti -infective drug, a cardiovascular (CV) system drug, a central nervous system (CNS) drug, an autonomic nervous system (ANS) drug, a respiratory tract drug, a gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid or electrolyte balance, a hematologic drug, an antineoplastic, an immunomodulation drug, an ophthalmic, otic or nasal drug, a topical drug, a nutritional drug or the like. Such drugs are well known in the art, including formulations, indications, dosing and administration for each presented herein (see, e.g., Nursing 2001 Handbook of Drugs, 21.sup.st edition, Springhouse Corp., Springhouse, Pa., 2001 ; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, N.J.; Pharmcotherapy Handbook, Wells et al., ed., Appleton & Lange, Stamford, Conn., each entirely incorporated herein by reference).

[00160] The anti-infective drug can be at least one selected from amebicides or at least one antiprotozoals, anthelmintics, antifungals, antimalarials, antituberculotics or at least one antileprotics, aminoglycosides, penicillins, cephalosporins, tetracyclines, sulfonamides, fluoroquinolones, antivirals, macrolide anti-infectives, and miscellaneous anti-infectives. The CV drug can be at least one selected from inotropics, antiarrhythmics, antianginals, antihypertensives, antilipemics, and miscellaneous cardiovascular drugs. The CNS drug can be at least one selected from normarcotic analgesics or at least one selected from antipyretics, nonsteroidal anti-inflammatory drugs, narcotic or at least one opioid analgesics, sedative- hypnotics, anticonvulsants, antidepressants, antianxiety drugs, antipsychotics, central nervous system stimulants, antiparkinsonians, and miscellaneous central nervous system drugs. The ANS drug can be at least one selected from cholinergics (parasympathomimetics), anticholinergics, adrenergics (sympathomimetics), adrenergic blockers (sympatholytics), skeletal muscle relaxants, and neuromuscular blockers. The respiratory tract drug can be at least one selected from antihistamines, bronchodilators, expectorants or at least one antitussive, and miscellaneous respiratory drugs. The GI tract drug can be at least one selected from antacids or at least one adsorbent or at least one antiflatulent, digestive enzyme or at least one gallstone solubilizer, antidiarrheals, laxatives, antiemetics, and antiulcer drugs. The hormonal drug can be at least one selected from corticosteroids, androgens or at least one anabolic steroid, estrogen or at least one progestin, gonadotropin, antidiabetic drug or at least one glucagon, thyroid hormone, thyroid hormone antagonist, pituitary hormone, and parathyroid-like drug. The drug for fluid and electrolyte balance can be at least one selected from diuretics, electrolytes or at least one replacement solution, acidifier or at least one alkalinizer. The hematologic drug can be at least one selected from hematinics, anticoagulants, blood derivatives, and thrombolytic enzymes. The antineoplastics can be at least one selected from alkylating drugs, antimetabolites, antibiotic antineoplastics, antineoplastics that alter hormone balance, and miscellaneous antineoplastics. The immunomodulation drug can be at least one selected from immunosuppressants, vaccines or at least one toxoid, antitoxin or at least one antivenin, immune serum, and biological response modifier. The ophthalmic, otic, and nasal drugs can be at least one selected from ophthalmic anti-infectives, ophthalmic anti-inflammatories, miotics, mydriatics, ophthalmic vasoconstrictors, miscellaneous ophthalmics, otics, and nasal drugs. The topical drug can be at least one selected from local anti-infectives, scabicides or at least one pediculicide or topical corticosteroid. The nutritional drug can be at least one selected from vitamins, minerals, or calorics. See, e.g., contents of Nursing 2001 Drug Handbook, supra. [00161] The at least one amebicide or antiprotozoal can be at least one selected from atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole hydrochloride, and pentamidine isethionate. The at least one anthelmintic can be at least one selected from mebendazole, pyrantel pamoate, and thiabendazole. The at least one antifungal can be at least one selected from amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposomal, fluconazole, flucytosine, griseofulvin microsize, griseofulvin ultramicrosize, itraconazole, ketoconazole, nystatin, and terbinafine hydrochloride. The at least one antimalarial can be at least one selected from chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine, and pyrimethamine with sulfadoxine. The at least one antituberculotic or antileprotic can be at least one selected from clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, and streptomycin sulfate. The at least one aminoglycoside can be at least one selected from amikacin sulfate, gentamicin sulfate, neomycin sulfate, streptomycin sulfate, and tobramycin sulfate. The at least one penicillin can be at least one selected from amoxcillin/clavulanate potassium, amoxicillin trihydrate, ampicillin, ampicillin sodium, ampicillin trihydrate, ampicillin sodium/sulbactam sodium, cloxacillin sodium, dicloxacillin sodium, mezlocillin sodium, nafcillin sodium, oxacillin sodium, penicillin G benzathine, penicillin G potassium, penicillin G procaine, penicillin G sodium, penicillin V potassium, piperacillin sodium, piperacillin sodium/tazobactam sodium, ticarcillin disodium, and ticarcillin disodium/clavulanate potassium.

[00162] The at least one cephalosporin can be at least one selected from cefaclor, cefadroxil, cefazolin sodium, cefdinir, cefepime hydrochloride, cefixime, cefinetazole sodium, cefonicid sodium, cefoperazone sodium, cefotaxime sodium, cefotetan disodium, cefoxitin sodium, cefpodoxime proxetil, cefprozil, ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium, cefuroxime axetil, cefuroxime sodium, cephalexin hydrochloride, cephalexin monohydrate, cephradine, and loracarbef. The at least one tetracycline can be at least one selected from demeclocycline hydrochloride, doxycycline calcium, doxycycline hyclate, doxycycline hydrochloride, doxycycline monohydrate, minocycline hydrochloride, and tetracycline hydrochloride. The at least one sulfonamide can be at least one selected from co- trimoxazole, sulfadiazine, sulfamethoxazole, sulfisoxazole, and sulfisoxazole acetyl. The at least one fluoroquinolone can be at least one selected from alatrofloxacin mesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, and trovafioxacin mesylate. The at least one fluoroquinolone can be at least one selected from alatrofloxacin mesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, and trovafioxacin mesylate. The at least one antiviral can be at least one selected from abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine, lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, and zidovudine. The at least one macroline anti-infective can be at least one selected from azithromycin, clarithromycin, dirithromycin, erythromycin base, erythromycin estolate, erythromycin ethylsuccinate, erythromycin lactobionate, and erythromycin stearate. The at least one miscellaneous anti-infective can be at least one selected from aztreonam, bacitracin, chloramphenicol sodium sucinate, clindamycin hydrochloride, clindamycin palmitate hydrochloride, clindamycin phosphate, imipenem and cilastatin sodium, meropenem, nitrofurantoin macrocrystals, nitrofurantoin microcrystals, quinupristin/dalfopristin, spectinomycin hydrochloride, trimethoprim, and vancomycin hydrochloride. (See, e.g., pp. 24- 214 of Nursing 2001 Drug Handbook.).

[00163] The at least one inotropic can be at least one selected from aminone lactate, digoxin, and milrinone lactate. The at least one antiarrhythmic can be at least one selected from adenosine, amiodarone hydrochloride, atropine sulfate, bretylium tosylate, diltiazem hydrochloride, disopyramide, disopyramide phosphate, esmolol hydrochloride, flecamide acetate, ibutilide fumarate, lidocaine hydrochloride, mexiletine hydrochloride, moricizine hydrochloride, phenyloin, phenyloin sodium, procainamide hydrochloride, propafenone hydrochloride, propranolol hydrochloride, quinidine bisulfate, quinidine gluconate, quinidine polygalacturonate, quinidine sulfate, sotalol, tocamide hydrochloride, and verapamil hydrochloride. The at least one antianginal can be at least one selected from amlodipidine besylate, amyl nitrite, bepridil hydrochloride, diltiazem hydrochloride, isosorbide dinitrate, isosorbide mononitrate, nadolol, nicardipine hydrochloride, nifedipine, nitroglycerin, propranolol hydrochloride, verapamil, and verapamil hydrochloride. The at least one antihypertensive can be at least one selected from acebutolol hydrochloride, amlodipine besylate, atenolol, benazepril hydrochloride, betaxolol hydrochloride, bisoprolol fumarate, candesartan cilexetil, captopril, carteolol hydrochloride, carvedilol, clonidine, clonidine hydrochloride, diazoxide, diltiazem hydrochloride, doxazosin mesylate, enalaprilat, enalapril maleate, eprosartan mesylate, felodipine, fenoldopam mesylate, fosinopril sodium, guanabenz acetate, guanadrel sulfate, guanfacine hydrochloride, hydralazine hydrochloride, irbesartan, isradipine, labetalol hydrochloride, lisinopril, losartan potassium, methyldopa, methyldopate hydrochloride, metoprolol succinate, metoprolol tartrate, minoxidil, moexipril hydrochloride, nadolol, nicardipine hydrochloride, nifedipine, nisoldipine, nitroprusside sodium, penbutolol sulfate, perindopril erbumine, phentolamine mesylate, pindolol, prazosin hydrochloride, propranolol hydrochloride, quinapril hydrochloride, ramipril, telmisartan, terazosin hydrochloride, timolol maleate, trandolapril, valsartan, and verapamil hydrochloride. The at least one antilipemic can be at least one selected from atorvastatin calcium, cerivastatin sodium, cholestyramine, colestipol hydrochloride, fenofibrate (micronized), fluvastatin sodium, gemfibrozil, lovastatin, niacin, pravastatin sodium, and simvastatin. The at least one miscellaneous CV drug can be at least one selected from abciximab, alprostadil, arbutamine hydrochloride, cilostazol, clopidogrel bisulfate, dipyridamole, eptifibatide, midodrine hydrochloride, pentoxifylline, ticlopidine hydrochloride, and tirofiban hydrochloride. (See, e.g., pp. 215-336 of Nursing 2001 Drug Handbook.).

[00164] The at least one normarcotic analgesic or antipyretic can be at least one selected from acetaminophen, aspirin, choline magnesium trisalicylate, diflunisal, and magnesium salicylate. The at least one nonsteroidal anti-inflammatory drug can be at least one selected from celecoxib, diclofenac potassium, diclofenac sodium, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen, indomethacin, indomethacin sodium trihydrate, ketoprofen, ketorolac tromethamine, nabumetone, naproxen, naproxen sodium, oxaprozin, piroxicam, rofecoxib, and sulindac. The at least one narcotic or opioid analgesic can be at least one selected from alfentanil hydrochloride, buprenorphine hydrochloride, butorphanol tartrate, codeine phosphate, codeine sulfate, fentanyl citrate, fentanyl transdermal system, fentanyl transmucosal, hydromorphone hydrochloride, meperidine hydrochloride, methadone hydrochloride, morphine hydrochloride, morphine sulfate, morphine tartrate, nalbuphine hydrochloride, oxycodone hydrochloride, oxycodone pectinate, oxymorphone hydrochloride, pentazocine hydrochloride, pentazocine hydrochloride and naloxone hydrochloride, pentazocine lactate, propoxyphene hydrochloride, propoxyphene napsylate, remifentanil hydrochloride, sufentanil citrate, and tramadol hydrochloride. The at least one sedative-hypnotic can be at least one selected from chloral hydrate, estazolam, flurazepam hydrochloride, pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbital sodium, temazepam, triazolam, zaleplon, and Zolpidem tartrate. The at least one anticonvulsant can be at least one selected from acetazolamide sodium, carbamazepine, clonazepam, clorazepate dipotassium, diazepam, divalproex sodium, ethosuximde, fosphenyloin sodium, gabapentin, lamotrigine, magnesium sulfate, phenobarbital, phenobarbital sodium, phenyloin, phenyloin sodium, phenyloin sodium (extended), primidone, tiagabine hydrochloride, topiramate, valproate sodium, and valproic acid. The at least one antidepressant can be at least one selected from amitriptyline hydrochloride, amitriptyline pamoate, amoxapine, bupropion hydrochloride, citalopram hydrobromide, clomipramine hydrochloride, desipramine hydrochloride, doxepin hydrochloride, fluoxetine hydrochloride, imipramine hydrochloride, imipramine pamoate, mirtazapine, nefazodone hydrochloride, nortriptyline hydrochloride, paroxetine hydrochloride, phenelzine sulfate, sertraline hydrochloride, tranylcypromine sulfate, trimipramine maleate, and venlafaxine hydrochloride. The at least one antianxiety drug can be at least one selected from alprazolam, buspirone hydrochloride, chlordiazepoxide, chlordiazepoxide hydrochloride, clorazepate dipotassium, diazepam, doxepin hydrochloride, hydroxyzine embonate, hydroxyzine hydrochloride, hydroxyzine pamoate, lorazepam, mephrobamate, midazolam hydrochloride, and oxazepam. The at least one antipsychotic drug can be at least one selected from chlorpromazine hydrochloride, clozapine, fluphenazine decanoate, fluephenazine enanthate, fluphenazine hydrochloride, haloperidol, haloperidol decanoate, haloperidol lactate, loxapine hydrochloride, loxapine succinate, mesoridazine besylate, molindone hydrochloride, olanzapine, perphenazine, pimozide, prochlorperazine, quetiapine fumarate, risperidone, thioridazine hydrochloride, thiothixene, thiothixene hydrochloride, and trifluoperazine hydrochloride. The at least one central nervous system stimulant can be at least one selected from amphetamine sulfate, caffeine, dextroamphetamine sulfate, doxapram hydrochloride, methamphetamine hydrochloride, methylphenidate hydrochloride, modafinil, pemoline, and phentermine hydrochloride. The at least one antiparkinsonian can be at least one selected from amantadine hydrochloride, benztropine mesylate, biperiden hydrochloride, biperiden lactate, bromocriptine mesylate, carbidopa-levodopa, entacapone, levodopa, pergolide mesylate, pramipexole dihydrochloride, ropinirole hydrochloride, selegiline hydrochloride, tolcapone, and trihexyphenidyl hydrochloride. The at least one miscellaneous central nervous system drug can be at least one selected from bupropion hydrochloride, donepezil hydrochloride, droperidol, fluvoxamine maleate, lithium carbonate, lithium citrate, naratriptan hydrochloride, nicotine polacrilex, nicotine transdermal system, propofol, rizatriptan benzoate, sibutramine hydrochloride monohydrate, sumatriptan succinate, tacrine hydrochloride, and zolmitriptan. (See, e.g., pp. 337- 530 of Nursing 2001 Drug Handbook.).

[00165] The at least one cholinergic (e.g., parasympathomimetic) can be at least one selected from bethanechol chloride, edrophonium chloride, neostigmine bromide, neostigmine methylsulfate, physostigmine salicylate, and pyridostigmine bromide. The at least one anticholinergic can be at least one selected from atropine sulfate, dicyclomine hydrochloride, glycopyrrolate, hyoscyamine, hyoscyamine sulfate, propantheline bromide, scopolamine, scopolamine butylbromide, and scopolamine hydrobromide. The at least one adrenergic (sympathomimetics) can be at least one selected from dobutamine hydrochloride, dopamine hydrochloride, metaraminol bitartrate, norepinephrine bitartrate, phenylephrine hydrochloride, pseudoephedrine hydrochloride, and pseudoephedrine sulfate. The at least one adrenergic blocker (sympatholytic) can be at least one selected from dihydroergotamine mesylate, ergotamine tartrate, methysergide maleate, and propranolol hydrochloride. The at least one skeletal muscle relaxant can be at least one selected from baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine hydrochloride, dantrolene sodium, methocarbamol, and tizanidine hydrochloride. The at least one neuromuscular blocker can be at least one selected from atracurium besylate, cisatracurium besylate, doxacurium chloride, mivacurium chloride, pancuronium bromide, pipecuronium bromide, rapacuronium bromide, rocuronium bromide, succinylcholine chloride, tubocurarine chloride, and vecuronium bromide. (See, e.g., pp. 531-84 of Nursing 2001 Drug Handbook.).

[00166] The at least one antihistamine can be at least one selected from brompheniramine maleate, cetirizine hydrochloride, chlorpheniramine maleate, clemastine fumarate, cyproheptadine hydrochloride, diphenhydramine hydrochloride, fexofenadine hydrochloride, loratadine, promethazine hydrochloride, promethazine theoclate, and triprolidine hydrochloride. The at least one bronchodilator can be at least one selected from albuterol, albuterol sulfate, aminophylline, atropine sulfate, ephedrine sulfate, epinephrine, epinephrine bitartrate, epinephrine hydrochloride, ipratropium bromide, isoproterenol, isoproterenol hydrochloride, isoproterenol sulfate, levalbuterol hydrochloride, metaproterenol sulfate, oxtriphylline, pirbuterol acetate, salmeterol xinafoate, terbutaline sulfate, and theophylline. The at least one expectorant or antitussive can be at least one selected from benzonatate, codeine phosphate, codeine sulfate, dextramethorphan hydrobromide, diphenhydramine hydrochloride, guaifenesin, and hydromorphone hydrochloride. The at least one miscellaneous respiratory drug can be at least one selected from acetylcysteine, beclomethasone dipropionate, beractant, budesonide, calfactant, cromolyn sodium, domase alfa, epoprostenol sodium, flunisolide, fluticasone propionate, montelukast sodium, nedocromil sodium, palivizumab, triamcinolone acetonide, zafirlukast, and zileuton. (See, e.g., pp. 585-642 of Nursing 2001 Drug Handbook.).

[00167] The at least one antacid, adsorbent, or antiflatulent can be at least one selected from aluminum carbonate, aluminum hydroxide, calcium carbonate, magaldrate, magnesium hydroxide, magnesium oxide, simethicone, and sodium bicarbonate. The at least one digestive enzyme or gallstone solubilizer can be at least one selected from pancreatin, pancrelipase, and ursodiol. The at least one antidiarrheal can be at least one selected from attapulgite, bismuth subsalicylate, calcium polycarbophil, diphenoxylate hydrochloride and atropine sulfate, loperamide, octreotide acetate, opium tincture, and opium tincure (camphorated). The at least one laxative can be at least one selected from bisocodyl, calcium polycarbophil, cascara sagrada, cascara sagrada aromatic fluidextract, cascara sagrada fluidextract, castor oil, docusate calcium, docusate sodium, glycerin, lactulose, magnesium citrate, magnesium hydroxide, magnesium sulfate, methylcellulose, mineral oil, polyethylene glycol or electrolyte solution, psyllium, senna, and sodium phosphates. The at least one antiemetic can be at least one selected from chlorpromazine hydrochloride, dimenhydrinate, dolasetron mesylate, dronabinol, granisetron hydrochloride, meclizine hydrochloride, metocloproamide hydrochloride, ondansetron hydrochloride, perphenazine, prochlorperazine, prochlorperazine edisylate, prochlorperazine maleate, promethazine hydrochloride, scopolamine, thiethylperazine maleate, and trimethobenzamide hydrochloride. The at least one antiulcer drug can be at least one selected from cimetidine, cimetidine hydrochloride, famotidine, lansoprazole, misoprostol, nizatidine, omeprazole, rabeprozole sodium, rantidine bismuth citrate, ranitidine hydrochloride, and sucralfate. (See, e.g., pp. 643-95 of Nursing 2001 Drug Handbook.).

[00168] The at least one coricosteroid can be at least one selected from betamethasone, betamethasone acetate or betamethasone sodium phosphate, betamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisone acetate, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, and triamcinolone diacetate. The at least one androgen or anabolic steroid can be at least one selected from danazol, fluoxymesterone, methyltestosterone, nandrolone decanoate, nandrolone phenpropionate, testosterone, testosterone cypionate, testosterone enanthate, testosterone propionate, and testosterone transdermal system. The at least one estrogen or progestin can be at least one selected from esterified estrogens, estradiol, estradiol cypionate, estradiol/norethindrone acetate transdermal system, estradiol valerate, estrogens (conjugated), estropipate, ethinyl estradiol, ethinyl estradiol and desogestrel, ethinyl estradiol and ethynodiol diacetate, ethinyl estradiol and desogestrel, ethinyl estradiol and ethynodiol diacetate, ethinyl estradiol and levonorgestrel, ethinyl estradiol and norethindrone, ethinyl estradiol and norethindrone acetate, ethinyl estradiol and norgestimate, ethinyl estradiol and norgestrel, ethinyl estradiol and norethindrone and acetate and ferrous fumarate, levonorgestrel, medroxyprogesterone acetate, mestranol and norethindron, norethindrone, norethindrone acetate, norgestrel, and progesterone. The at least one gonadroptropin can be at least one selected from ganirelix acetate, gonadoreline acetate, histrelin acetate, and menotropins. The at least one antidiabetic or glucaon can be at least one selected from acarbose, chlorpropamide, glimepiride, glipizide, glucagon, glyburide, insulins, metformin hydrochloride, miglitol, pioglitazone hydrochloride, repaglinide, rosiglitazone maleate, and troglitazone. The at least one thyroid hormone can be at least one selected from levothyroxine sodium, liothyronine sodium, liotrix, and thyroid. The at least one thyroid hormone antagonist can be at least one selected from methimazole, potassium iodide, potassium iodide (saturated solution), propylthiouracil, radioactive iodine (sodium iodide .sup.13 II), and strong iodine solution. The at least one pituitary hormone can be at least one selected from corticotropin, cosyntropin, desmophressin acetate, leuprolide acetate, repository corticotropin, somatrem, somatropin, and vasopressin. The at least one parathyroid-like drug can be at least one selected from calcifediol, calcitonin (human), calcitonin (salmon), calcitriol, dihydrotachysterol, and etidronate disodium. (See, e.g., pp. 696-796 of Nursing 2001 Drug Handbook.). [00169] The at least one diuretic can be at least one selected from acetazolamide, acetazolamide sodium, amiloride hydrochloride, bumetanide, chlorthalidone, ethacrynate sodium, ethacrynic acid, furosemide, hydrochlorothiazide, indapamide, mannitol, metolazone, spironolactone, torsemide, triamterene, and urea. The at least one electrolyte or replacement solution can be at least one selected from calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, calcium lactate, calcium phosphate (dibasic), calcium phosphate (tribasic), dextran (high-molecular-weight), dextran (low-molecular-weight), hetastarch, magnesium chloride, magnesium sulfate, potassium acetate, potassium bicarbonate, potassium chloride, potassium gluconate, Ringer's injection, Ringer's injection (lactated), and sodium chloride. The at least one acidifier or alkalinizer can be at least one selected from sodium bicarbonate, sodium lactate, and tromethamine. (See, e.g., pp. 797-833 of Nursing 2001 Drug Handbook.).

[00170] The at least one hematinic can be at least one selected from ferrous fumarate, ferrous gluconate, ferrous sulfate, ferrous sulfate (dried), iron dextran, iron sorbitol, polysaccharide-iron complex, and sodium ferric gluconate complex. The at least one anticoagulant can be at least one selected from ardeparin sodium, dalteparin sodium, danaparoid sodium, enoxaparin sodium, heparin calcium, heparin sodium, and warfarin sodium. The at least one blood derivative can be at least one selected from albumin 5%, albumin 25%, antihemophilic factor, anti-inhibitor coagulant complex, antithrombin III (human), factor IX (human), factor IX complex, and plasma protein fractions. The at least one thrombolytic enzyme can be at least one selected from alteplase, anistreplase, reteplase (recombinant), streptokinase, and urokinase. (See, e.g., pp. 834-66 of Nursing 2001 Drug Handbook.).

[00171] The at least one alkylating drug can be at least one selected from busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, ifosfamide, lomustine, mechlorethamine hydrochloride, melphalan, melphalan hydrochloride, streptozocin, temozolomide, and thiotepa. The at least one antimetabolite can be at least one selected from capecitabine, cladribine, cytarabine, floxuridine, fludarabine phosphate, fluorouracil, hydroxyurea, mercaptopurine, methotrexate, methotrexate sodium, and thioguanine. The at least one antibiotic antineoplastic can be at least one selected from bleomycin sulfate, dactinomycin, daunorubicin citrate liposomal, daunorubicin hydrochloride, doxorubicin hydrochloride, doxorubicin hydrochloride liposomal, epirubicin hydrochloride, idarubicin hydrochloride, mitomycin, pentostatin, plicamycin, and valrubicin. The at least one antineoplastic that alters hormone balance can be at least one selected from anastrozole, bicalutamide, estramustine phosphate sodium, exemestane, flutamide, goserelin acetate, letrozole, leuprolide acetate, megestrol acetate, nilutamide, tamoxifen citrate, testolactone, and toremifene citrate. The at least one miscellaneous antineoplastic can be at least one selected from asparaginase, bacillus Calmette-Guerin (BCG) (live intravesical), dacarbazine, docetaxel, etoposide, etoposide phosphate, gemcitabine hydrochloride, irinotecan hydrochloride, mitotane, mitoxantrone hydrochloride, paclitaxel, pegaspargase, porfimer sodium, procarbazine hydrochloride, rituximab, teniposide, topotecan hydrochloride, trastuzumab, tretinoin, vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate. (See, e.g., pp. 867-963 of Nursing 2001 Drug Handbook.).

[00172] The at least one immunosuppressant can be at least one selected from azathioprine, basiliximab, cyclosporine, daclizumab, lymphocyte immune globulin, muromonab- CD3, mycophenolate mofetil, mycophenolate mofetil hydrochloride, sirolimus, and tacrolimus. The at least one vaccine or toxoid can be at least one selected from BCG vaccine, cholera vaccine, diphtheria and tetanus toxoids (adsorbed), diphtheria and tetanus toxoids and acellular pertussis vaccine adsorbed, diphtheria and tetanus toxoids and whole-cell pertussis vaccine, Haemophilus b conjugate vaccines, hepatitis A vaccine (inactivated), hepatitis B vaccine (recombinant), influenza virus vaccine 1999-2000 trivalent types A & B (purified surface antigen), influenza virus vaccine 1999-2000 trivalent types A & B (subvirion or purified subvirion), influenza virus vaccine 1999-2000 trivalent types A & B (whole virion), Japanese encephalitis virus vaccine (inactivated), Lyme disease vaccine (recombinant OspA), measles and mumps and rubella virus vaccine (live), measles and mumps and rubella virus vaccine (live attenuated), measles virus vaccine (live attenuated), meningococcal polysaccharide vaccine, mumps virus vaccine (live), plague vaccine, pneumococcal vaccine (polyvalent), poliovirus vaccine (inactivated), poliovirus vaccine (live, oral, trivalent), rabies vaccine (adsorbed), rabies vaccine (human diploid cell), rubella and mumps virus vaccine (live), rubella virus vaccine (live, attenuated), tetanus toxoid (adsorbed), tetanus toxoid (fluid), typhoid vaccine (oral), typhoid vaccine (parenteral), typhoid Vi polysaccharide vaccine, varicella virus vaccine, and yellow fever vaccine. The at least one antitoxin or antivenin can be at least one selected from black widow spider antivenin, Crotalidae antivenom (polyvalent), diphtheria antitoxin (equine), and Micrurus fulvius antivenin. The at least one immune serum can be at least one selected from cytomegalovirus immune globulin (intraveneous), hepatitis B immune globulin (human), immune globulin intramuscular, immune globulin intravenous, rabies immune globulin (human), respiratory syncytial virus immune globulin intravenous (human), Rh.sub.O(D) immune globulin (human), Rh.sub.O(D) immune globulin intravenous (human), tetanus immune globulin (human), and varicella-zoster immune globulin. The at least one biological response modifier can be at least one selected from aldesleukin, epoetin alfa, filgrastim, glatiramer acetate for injection, interferon alfacon-1, interferon alfa-2a (recombinant), interferon alfa-2b (recombinant), interferon beta- la, interferon beta- lb (recombinant), interferon gamma- lb, levamisole hydrochloride, oprelvekin, and sargramostim. (See, e.g., pp. 964-1040 of Nursing 2001 Drug Handbook.).

[00173] The at least one ophthalmic anti-infective can be selected form bacitracin, chloramphenicol, ciprofloxacin hydrochloride, erythromycin, gentamicin sulfate, ofloxacin 0.3%, polymyxin B sulfate, sulfacetamide sodium 10%, sulfacetamide sodium 15%), sulfacetamide sodium 30%, tobramycin, and vidarabine. The at least one ophthalmic antiinflammatory can be at least one selected from dexamethasone, dexamethasone sodium phosphate, diclofenac sodium 0.1 %, fluorometholone, flurbiprofen sodium, ketorolac tromethamine, prednisolone acetate (suspension) and prednisolone sodium phosphate (solution). The at least one miotic can be at least one selected from acetylcholine chloride, carbachol (intraocular), carbachol (topical), echothiophate iodide, pilocarpine, pilocarpine hydrochloride, and pilocarpine nitrate. The at least one mydriatic can be at least one selected from atropine sulfate, cyclopentolate hydrochloride, epinephrine hydrochloride, epinephryl borate, homatropine hydrobromide, phenylephrine hydrochloride, scopolamine hydrobromide, and tropicamide. The at least one ophthalmic vasoconstrictor can be at least one selected from naphazoline hydrochloride, oxymetazoline hydrochloride, and tetrahydrozoline hydrochloride. The at least one miscellaneous ophthalmic can be at least one selected from apraclonidine hydrochloride, betaxolol hydrochloride, brimonidine tartrate, carteolol hydrochloride, dipivefrin hydrochloride, dorzolamide hydrochloride, emedastine difumarate, fluorescein sodium, ketotifen fumarate, latanoprost, levobunolol hydrochloride, metipranolol hydrochloride, sodium chloride (hypertonic), and timolol maleate. The at least one otic can be at least one selected from boric acid, carbamide peroxide, chloramphenicol, and triethanolamine polypeptide oleate-condensate. The at least one nasal drug can be at least one selected from beclomethasone dipropionate, budesonide, ephedrine sulfate, epinephrine hydrochloride, flunisolide, fluticasone propionate, naphazoline hydrochloride, oxymetazoline hydrochloride, phenylephrine hydrochloride, tetrahydrozoline hydrochloride, triamcinolone acetonide, and xylometazoline hydrochloride. (See, e.g., pp. 1041-97 of Nursing 2001 Drug Handbook.).

[00174] The at least one local anti-infective can be at least one selected from acyclovir, amphotericin B, azelaic acid cream, bacitracin, butoconazole nitrate, clindamycin phosphate, clotrimazole, econazole nitrate, erythromycin, gentamicin sulfate, ketoconazole, mafenide acetate, metronidazole (topical), miconazole nitrate, mupirocin, naftifine hydrochloride, neomycin sulfate, nitrofurazone, nystatin, silver sulfadiazine, terbinafme hydrochloride, terconazole, tetracycline hydrochloride, tioconazole, and tolnaftate. The at least one scabicide or pediculicide can be at least one selected from crotamiton, lindane, permethrin, and pyrethrins. The at least one topical corticosteroid can be at least one selected from betamethasone dipropionate, betamethasone valerate, clobetasol propionate, desonide, desoximetasone, dexamethasone, dexamethasone sodium phosphate, diflorasone diacetate, fluocinolone acetonide, fluocinonide, flurandrenolide, fluticasone propionate, halcionide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocorisone valerate, mometasone furoate, and triamcinolone acetonide. (See, e.g., pp. 1098-1 136 of Nursing 2001 Drug Handbook.).

[00175] The at least one vitamin or mineral can be at least one selected from vitamin A, vitamin B complex, cyanocobalamin, folic acid, hydroxocobalamin, leucovorin calcium, niacin, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin C, vitamin D, cholecalciferol, ergocalciferol, vitamin D analogue, doxercalciferol, paricalcitol, vitamin E, vitamin K analogue, phytonadione, sodium fluoride, sodium fluoride (topical), trace elements, chromium, copper, iodine, manganese, selenium, and zinc. The at least one caloric can be at least one selected from amino acid infusions (crystalline), amino acid infusions in dextrose, amino acid infusions with electrolytes, amino acid infusions with electrolytes in dextrose, amino acid infusions for hepatic failure, amino acid infusions for high metabolic stress, amino acid infusions for renal failure, dextrose, fat emulsions, and medium-chain triglycerides. (See, e.g., pp. 1137-63 of Nursing 2001 Drug Handbook.).

[00176] The compositions provided by the present invention can further comprise at least one of any suitable and effective amount of a composition or pharmaceutical composition comprising at least one of an anti-IL-12 antibody contacted or administered to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy, a TNF antagonist (e.g., but not limited to a TNF chemical or protein antagonist, TNF monoclonal or polyclonal antibody or fragment, a soluble TNF receptor (e.g., p55, p70 or p85) or fragment, fusion polypeptides thereof, or a small molecule TNF antagonist, e.g., TNF binding protein I or II (TBP-1 or TBP-II), nerelimonmab, infliximab, etemacept, CDP-571, CDP-870, afelimomab, lenercept, and the like), an antirheumatic (e.g., methotrexate, auranofm, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a fluororquinolone, a macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic, a corticosteriod, an anabolic steroid, a diabetes related agent, a mineral, a nutritional, a thyroid agent, a vitamin, a calcium related hormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer, a laxative, an anticoagulant, an erythropoietin (e.g., epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin, an immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), a growth hormone, a hormone replacement drug, an estrogen receptor modulator, a mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or a cytokine antagonist. Non-limiting examples of such cytokines include, but are not limited to, any of IL-1 to IL-23 (e.g., IL-1, IL-2, etc.). Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2.sup.nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), each of which references are entirely incorporated herein by reference.

[00177] Such anti-cancer or anti-infectives can also include toxin molecules that are associated, bound, co-formulated or co-administered with at least one antibody of the present invention. The toxin can optionally act to selectively kill the pathologic cell or tissue. The pathologic cell can be a cancer or other cell. Such toxins can be, but are not limited to, purified or recombinant toxin or toxin fragment comprising at least one functional cytotoxic domain of toxin, e.g., selected from at least one of ricin, diphtheria toxin, a venom toxin, or a bacterial toxin. The term toxin also includes both endotoxins and exotoxins produced by any naturally occurring, mutant or recombinant bacteria or viruses which may cause any pathological condition in humans and other mammals, including toxin shock, which can result in death. Such toxins may include, but are not limited to, enterotoxigenic E. coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas enterotoxins, toxic shock syndrome toxin- 1 (TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal enterotoxins and the like. Such bacteria include, but are not limited to, strains of a species of enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7), Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcus pyogenes), Shigella species (e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei), Salmonella species (e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella enteritidis), Clostridium species (e.g., Clostridium perfringens, Clostridium dificile, Clostridium botulinum), Camphlobacter species (e.g., Camphlobacter jejuni, Camphlobacter fetus), Heliobacter species, (e.g., Heliobacter pylori), Aeromonas species (e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides, Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae, Vibrios parahemolyticus), Klebsiella species, Pseudomonas aeruginosa, and Streptococci. See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp 1-13, Little, Brown and Co., Boston, (1990); Evans et al., eds., Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed., pp 239-254, Plenum Medical Book Co., New York (1991); Mandell et al, Principles and Practice of Infectious Diseases, 3d. Ed., Churchill Livingstone, New York (1990); Berkow et al, eds., The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMS Microbiology Immunology, 76: 121-134 (1991); Marrack et al, Science, 248:705- 71 1 (1990), the contents of which references are incorporated entirely herein by reference.

[00178] In another aspect, the invention provides an isolated nucleic acid encoding a polypeptide having at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in sequence identity to any of the nucleic acids disclosed in SEQ ID NOS: 9, 10, 1 1 , or 12. In certain embodiments, the description provides isolated nucleic acid molecules that hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein- coding sequence of an IL-7 or HGF-β nucleic acid sequence, respectively. The invention also includes an isolated nucleic acid that encodes an an IL-7 or HGF-β polypeptide, or a fragment, homolog, analog, fusion protein, pseudopeptide, peptidomimetic or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in sequence identity to a polypeptide of SEQ ID NOS:3, 4, 5, 6, 7, 8, 13, or 14.

[00179] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91 : 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[00180] The description also provides substantially purified IL-7/HGF-P hybrid cytokine polypeptides or a functional portion thereof. In certain embodiments, an isolated, purified IL- 7/HGF"P hybrid cytokine polypeptide comprises at least one amino acid sequence that is substantially identical to an amino acid sequence as set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 13, or 14.

[00181] As described supra, the invention also provides chimeric or fusion proteins. As used herein, a "chimeric protein" or "fusion protein" comprises a polypeptide operatively-linked to another polypeptide, for example, one or more of the polypeptides chosen from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 13, or 14, or portions thereof. Within the fusion protein the polypeptide can correspond to all or a portion of a polypeptide chosen from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 13, or 14. In one embodiment, the fusion protein comprises at least one biologically active portion of the protein chosen from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 13, or 14. Within the chimera or fusion protein, the discrete polypeptides are fused in-frame with one another at the N-terminus or C- terminus. [00182] In certain instances, it may be desirable to immobilize the chimeric polypeptides of the invention to facilitate separation of the proteins. Thus, the description provides an hybrid cytokine fusion protein which includes a domain that allows the proteins to be bound to a solid state or matrix. For example, glutathione-S-transferase fusion proteins or conjugation of biotin and streptavidin. In one embodiment, the fusion protein is a GST- fusion protein in which the polypeptide sequences are fused to the C-terminus or N-terminus of the GST (glutathione S- transferase) sequences. In another embodiment, the fusion protein contains a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion can be increased through use of a heterologous signal sequence. In yet another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptides or polypeptide complex of as described herein is fused to sequences derived from a member of the immunoglobulin protein family. In one embodiment, the immunoglobulin fusion protein is incorporated into a pharmaceutical composition and administered to a subject to modulate an interaction between a ligand and a protein on the surface of a cell. The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand. Inhibition of the ligand interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction.

[00183] A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in- frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). One or more of SEQ ID NOs: 9-12, or portions thereof, can be cloned into such an expression vector such that the fusion moiety is linked in- frame to the desired polypeptide.

[00184] Antibodies

[00185] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen, comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab' and F(ab')2 fragments, and an Fab expression library. The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGj, IgG₂, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.

[00186] Antibodies can be prepared from the intact polypeptide or fragments containing peptides of interest as the immunizing agent. A preferred antigenic polypeptide fragment is 15- 100 contiguous amino acids of SEQ ID NOs: 3-8, 13, or 14. In certain embodiments, the present invention comprises antibodies that recognize and are specific for one or more epitopes of any of SEQ ID NOs: 3-8, 13, or 14, variants, portions and/or combinations thereof. In other embodiments, the antibodies of the invention may be specific for the cytokine receptor complex itself. In still other embodiments an antibody specific for the chimeric cytokine of the invention may function as the "receptor" - i.e., functioning in a transpresentation mechanism. In alternative embodiments antibodies of the invention may target and interfere with the chimeric cytokine/receptor interaction to inhibit signaling.

[00187] The preparation of polyclonal antibodies is well known in the molecular biology art; see for example, Production of Polyclonal Antisera in Immunochemical Processes (Manson, ed.), pages 1-5 (Humana Press 1992) and Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters in Current Protocols in Immunology, section 2.4.1 (1992). The preparation of monoclonal antibodies is also well known in the art; see for example, Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. 1988).

[00188] Monoclonal antibodies can be obtained by injecting mice or rabbits with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by techniques well known in the art.

[00189] In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods. Phage display and combinatorial methods can be used to isolate recombinant antibodies that bind to SEQ ID NOs: 5-12 or fragments thereof (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271 ; Winter et al. International Publication WO 92/20791 ; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246: 1275-1281 ; Griffths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982). [00190] Human monoclonal antibodies can also be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741 ; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7: 13-21 ; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81 :6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21 : 1323-1326).

[00191] A therapeutically useful antibody to the components of the complex of the invention or the complex itself may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, then substituting human residues into the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with immunogenicity of murine constant regions. Techniques for producing humanized monoclonal antibodies can be found in Jones et al., Nature 321 : 522, 1986 and Singer et al., J. Immunol. 150: 2844, 1993. The antibodies can also be derived from human antibody fragments isolated from a combinatorial immunoglobulin library; see, for example, Barbas et al., Methods: A Companion to Methods in Enzymology 2, 1 19, 1991.

[00192] In addition, chimeric antibodies can be obtained by splicing the genes from a mouse antibody molecule with appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological specificity; see, for example, Takeda et al., Nature 314: 544-546, 1985. A chimeric antibody is one in which different portions are derived from different animal species.

[00193] Anti-idiotype technology can be used to produce monoclonal antibodies that mimic an epitope. An anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the "image" of the epitope bound by the first monoclonal antibody. Alternatively, techniques used to produce single chain antibodies can be used to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Antibody fragments that recognize specific epitopes, e.g., extracellular epitopes, can be generated by techniques well known in the art. Such fragments include Fab fragments produced by proteolytic digestion, and Fab fragments generated by reducing disulfide bridges. When used for immunotherapy, the monoclonal antibodies, fragments thereof, or both may be unlabelled or labeled with a therapeutic agent. These agents can be coupled directly or indirectly to the monoclonal antibody by techniques well known in the art, and include such agents as drugs, radioisotopes, lectins and toxins.

[00194] The dosage ranges for the administration of monoclonal antibodies are large enough to produce the desired effect, and will vary with age, condition, weight, sex, age and the extent of the condition to be treated, and can readily be determined by one skilled in the art. Dosages can be about 0.1 mg/kg to about 2000 mg/kg. The monoclonal antibodies can be administered intravenously, intraperitoneally, intramuscularly, and/or subcutaneously.

[00195] In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of SEQ ID NOs: 3-8, 13, or 14 that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the protein sequence will indicate which regions of a polypeptide are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated herein by reference in their entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein. A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.

[00196] Human Antibodies

[00197] Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see ozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[00198] In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10:779-783 (1992)); Lonberg et al. (Nature 368:856-859 (1994)); Morrison (Nature 368:812-13 (1994)); Fishwild et al,(Nature Biotechnology 14:845-51 (1996)); Neuberger (Nature Biotechnology 14:826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13:65-93 (1995)).

[00199] Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.

[00200] Fab Fragments and Single Chain Antibodies

[00201] According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.

[00202] Bispecific Antibodies

[00203] Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.

[00204] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[00205] Antibody variable domains with the desired binding specificities (antibody- antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121-210 (1986).

[00206] According to another approach described in WO 96/2701 1, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[00207] Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[00208] Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[00209] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547- 1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al, Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[00210] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcganimaR), such as FcgammaRI (CD64), FcgammaRII (CD32) and FcgammaRIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.

[00211] Heteroconjugate Antibodies

[00212] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

[00213] Immunoconjugates

[00214] The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4- dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon- 14-labeled 1-isothiocyanatobenzy 1-3 -methyldi ethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.

[00215] In another embodiment, the antibody can be conjugated to a "receptor" for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" that is in turn conjugated to a cytotoxic agent.

[00216] A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight.-Common dosing frequencies may range, for example, from twice daily to once a week.

[00217] Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[00218] The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymefhylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[00219] Sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[00220] ELISA Assay

[00221] An agent for detecting an analyte protein is an antibody capable of binding to an analyte protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab)2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end- labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and "Practice and Thory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-an analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques intracavity, or transdermally, alone or with effector cells.

[00222] The nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, lupus erythematosus, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, leukemia, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, rheumatoid and osteoarthritis, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions of the like.

[00223] Preparations for administration of the therapeutic species described herein include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles including fluid and nutrient replenishers, electrolyte replenishers, and the like. Preservatives and other additives may be added such as, for example, antimicrobial agents, anti-oxidants, chelating agents and inert gases and the like. [00224] As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[00225] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, intraperitoneal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[00226] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor™. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00227] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., the therapeutic complex of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00228] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[00229] For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.

[00230] Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethan- e, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use. The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[00231] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[00232] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[00233] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,81 1. [00234] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[00235] A therapeutically effective dose refers to that amount of the therapeutic complex sufficient to result in amelioration or delay of symptoms. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[00236] Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, intravenous, intraperitoneal, parenteral or rectal administration.

[00237] Also disclosed according to the present invention is a kit or system utilizing any one of the methods, selection strategies, materials, or components described herein. Exemplary kits according to the present disclosure will optionally, additionally include instructions for performing methods or assays, packaging materials, one or more containers which contain an assay, a device or system components, or the like.

[00238] In an additional aspect, the present invention provides kits embodying the complex and methods of using disclosed herein. Kits of the invention optionally include one or more of the following: (1) polypeptide or nucleic acid components described herein; (2) instructions for practicing the methods described herein, and/or for operating the selection procedure herein; (3) one or more detection assay components; (4) a container for holding nucleic acids or polypeptides, other nucleic acids, transgenic plants, animals, cells, or the like and, (5) packaging materials.

[00239] The following examples and accompanying figures are shown and presented in order to make certain aspects of the present invention more clearly understood and are not intended to limit the scope of the invention described herein in any manner.

[00240] EXAMPLE 1

[00241] Growth of Pre-Pro-B, Pro-B and Pre-B Cell Compartments on Different Culture Media. As illustrated in FIGS. 1A and IB, after inoculation with freshly-harvested BM cells the pre-pro-B cell and pro-B cell compartments in the pro-B cell culture previously described by the inventors (Hayashi, et al., J. Exp. Med. 160: 1622-1639 (1984)) progressively expands with time, whereas the pre-B cell compartment progressively contracts. In contrast, under Whitlock/Witte-type culture conditions, the pre-B cell compartment progressively expands with time (FIGS. 1C and ID) whereas the pre-pro-B cell and pro-B cell compartments progressively contract (after a brief period of expansion). Furthermore, pre-pro-B cells and pro-B cells from the lymphoid culture system generate pre-B cells when placed in Whitlock/Witte cultures or CM therefrom.

[00242] EXAMPLE 2 [00243] Recreation of Bone Marrow Microenvironment for Early Lymphopoiesis In

Vitro. The nature of the interactions between BM lymphoid precursor cells and BM adherent microenvironmental cells was investigated by a combination of immunophenotyping and scanning and transmission electron microscopy of primary cultures.

[00244] The results of such studies show that two distinct microenvironmental regions are represented within the BM adherent cell layer: (a) paucilayer (PL) regions, composed of two or three horizontally oriented layers of stromal cells; and (b) multilayer (ML) regions, containing 4 to 8 layers of stromal cells. In both regions, proliferating lymphoid cells expressing the B220, and/or heat stable antigen (HSA) early B-lineage antigens, are "sandwiched" between adjacent layers of stromal cells and enveloped by cytoplasmic processes from interdigitating mouse macrophages (pseudoemperipolesis). Small clusters containing 5 to 50 lymphoid cells, preferentially develop in the PL regions are comprised primarily of TdT cells that can generate TdT.sup.+ cells upon transfer onto fresh adherent cells layers.

[00245] Under ideal conditions, individual clusters are clonally derived and the seeding efficiency of the culture system approaches 100%. Large clusters, containing up to 1,000 lymphoid cells, preferentially develop in the ML regions and are comprised primarily of TdT.sup.+ cells. The ML regions bear a close resemblance to the recently described pro-B cell- enriched, multi-cellular aggregate fraction of freshly harvested mouse BM. Hence, this system appears to structurally recreate in vitro the in vivo microenvironment for the development of pre- pro-B cells and pro-B cells.

[00246] EXAMPLE 3

[00247] Properties and Developmental Relationships of the Lymphoid Cells in the Adherent and Nonadherent Compartments of the Pro-B Culture System. Turning to FIG. 2, four sequentially appearing subsets of B-cell progenitors in the inventors' pro-B culture system were characterized. The first lymphoid subset consists of adherent TdT.sup.-; (early) pre-pro-B cells that reach plateau numbers on day 3; and the second subset consists of adherent TdT+/- (late) pre-pro-B cells that plateau on day 7. This is closely followed by a parallel increase in the number of TdT+ (early) and TdT- (late) pro-B cells in the non-adherent phase.

[00248] In vitro transfer experiments demonstrated that virtually all of the early pre-pro-B cells in freshly harvested rat BM adhere to the mouse BM stromal cells during the first 24 hrs of culture; and by day 7 of culture, these cells had increased more than 20-fold on a per cell basis and more than 70-fold on a per well basis (FIG. 3). It was also observed that a decrease in the concentration of fetal bovine serum in the culture medium resulted in the selective release of late, but not early, pre-pro-B cells into the non-adherent compartment. These studies indicate that a stepwise progression of the earliest detectable stages in lymphoid development was associated with changes in stromal/lymphoid cell interactions partly regulated by serum-dependent adhesion mechanisms.

[00249] EXAMPLE 4

[00250] Ability of Medium Conditioned by BM Stromal Cells to Selectively Support the Development of Pre-Pro-B Cells and Pro-B Cells in Vitro. Although the pro-B culture system is characterized by physical interaction-between developing lymphocytes, BM stromal cells and macrophages, experiments using microporous membrane culture inserts demonstrate that these cognitive recognition events, albeit more efficient, are not essential (FIG. 4). Similarly, medium conditioned (CM) with mouse BM adherent cells supported the proliferation of lymphoid precursor cells in a dose-dependent manner. Upon ultrafiltration, all of the lymphostimulatory activity in the CM was recovered in the 50-100 kD apparent MW fraction; and double immunofluorescence for incorporated bromodeoxyuridine (BrdU) and early B- lineage markers indicated that the lymphoproliferative response selectively involved early (TdT-) and late (TdT+) pre-pro-B cells, but not pro-B cells.

[00251] EXAMPLE 5

[00252] The Pre-pro-B Cell Growth-Stimulating Factor (PPBSF), Detection of IL-7 (SEQ ID NO:7, 8) and a Non-IL-7 Component of PPBSF. Inasmuch as IL-7 (SEQ ID NO:7, 8) is one of the cytokines most closely associated with early B-lineage development, BM adherent cells and stromal cell lines from wild-type and IL-7 gene-deleted (-/-) mice were utilized to investigate its possible regulatory role in the pro-B culture system. The results show that both rIL-7 and IL-7(-/-) CM maintain the viability of pre-pro-B cells from freshly harvested rat BM, but that neither induces them to proliferate and/or differentiate, even in the presence of IL-3, rSCF and/or rIGF. However, as seen in FIG. 5A (primary culture) when added to IL-7(-/-) CM, rIL-7 efficiently stimulates proliferation and differentiation of freshly harvested pre-pro-B cells. Conversely, anti-IL-7 mAb inhibits the expansion of pre-pro-B cells in culture, and adsorbs the pre-pro-B cell growth-stimulating activity from both IL-7(+/+) CM and rIL-7-supplemented IL-7(-/-) CM. Yet, anti-IL-7 mAb does not neutralize the pre-pro-B cell growth-stimulating activity of these CM; and rIL-7 does not restore this activity to anti-IL-7 mAb-adsorbed CM. These results suggest that the pre-pro-B cell growth-stimulating activity in the pro-B culture system is the property of a self-aggregating molecular complex of IL-7 and a second BM stromal cell-derived co-factor (See FIG. 6). The results also suggest that this pre-pro-B cell growth- stimulating factor (PPBSF) not only selectively stimulates proliferation of pre-pro-B cells, but "primes" them and/or their immediate descendants to respond directly to monomeric IL-7 (FIG. 5B, secondary culture).

[00253] EXAMPLE 6

[00254] PPBSF is a Covalently-linked Heterodimer of IL-7 (SEQ ID NO:7, 8) and an M_r 30,000 Dalton Co-factor. Direct evidence for the existence of PPBSF in CM generated under pro-B cell, but not pre-B cell, culture conditions is provided by Western immunoblot analysis (FIG. 7). The results demonstrate that, when developed with anti-IL-7 mAb, PPBSF migrates electrophoretically as an apparent 55 kD molecule under non-reducing conditions, whereas the IL-7 (SEQ ID NO:7, 8) component migrates as an apparent 25 kD molecule under reducing conditions. Furthermore, IL-7 exists almost entirely as an heterodimer (i.e. PPBSF) in pro-B-type cultures, and as a monomer in pre-B-type cultures. However, addition of rIL-7 or native IL-7 (from pre-B CM) to CM from IL-7 KO mice results in the rapid formation of apparent 45 kD (FIG. 8, lane 5) and 55 kD (FIG. 9, lane 3) molecular complexes, respectively, both of which have the functional properties of PPBSF.

[00255] Using a neutralizing antiserum prepared in IL-7(-/-) mice against IL-7(+/+) CM (and subsequently monoclonal antibody (IgG2a; clone A4) against affinity-purified PPBSF), the non-IL-7 component of PPBSF was identified by Western analysis as an apparent 30 kD molecule (FIG. 10, lanes 2 and 4; and FIG. 11). These Abs react with PPBSF-coF in both its heterodimeric and monomeric forms, and neutralize the PPBSF activity in CM (FIG. 12). PPBSF-coF is able to maintain the viability of pre-pro-B cells, but does not stimulate their proliferation unless complexed with IL-7 (SEQ ID NO:7, 8). It is constitutively produced by lines of IL-7(-/-) BM stromal cells under pro-B, but not pre-B-type culture conditions. It does not appear to be SCF, IGF-1 , TSLP, Fet3, SDF-I or the soluble form of the IL-7R.

[00256] EXAMPLE 7

[00257] Determination that PPBSF is a Hybrid Cytokine of IL-7 (SEQ ID NO: 7, 8) and the beta-chain of Hepatocyte Growth Factor/scatter Factor (HGF/SF) (SEQ ID NO:3, 4, 13, and 14); Identity of PPBSF-coF. To identify the PPBSF-coF, affinity purified native PPBSF was electrophoresed under reducing conditions and the 30 kDa band was subjected to amino acid analysis. The results demonstrated that the first 15 of 17 amino acid residues were identical to the published sequence of mouse HGF-beta chain (SEQ ID NO:3, 4) (FIG. 13), as was the overall molecular mass of the peptide. The identity of PPBSF-coF as the HFG-beta chain was confirmed by reciprocal Western blot analyses, in which antibodies to HGF-beta-chain reacted with purified native PPBSF-coF, and mAbs to PPBSF-coF reacted with rHGF. In addition, both anti-HGF and anti-HGF-beta antibodies neutralized the PPBSF activity in IL-7 (+/+) CM.

[00258] EXAMPLE 8

[00259] Identification and Cloning of a Variant of HGF mRNA. Total RNA was isolated with TRI_zoi_tm Reagent (Total RNA Isolation Reagent, Life Technologies, Gaithersburg, Md.) from IL-7 (-/-) BM stomal cells. Random-primed first-strand cDNA was generated from this RNA using MMLV reverse transcriptase (RETRO Script™, Amibion, Austin, Tex.). PCR reactions were performed with the cDNA, Taq polymerase (Life Technologies, Gaithersburg, Md.), and primers designed to amplify the entire coding sequence of mouse HGF: 5'- C AGTCTGCTCGAACTGC A-3 ' (SEQ ID NO: l) (in 5'flanking region) 5'- TGGCCTCTTCT ATGGCTA-3 ' (SEQ ID NO:2) (in 3' flanking region). Two RT-PCR products were obtained when the amplified fragments were separated on 1% agarose gel and visualized by ethidium bromide (FIG. 16). One of these products corresponded to the full-length HGF cDNA (2230 bp). However, the second product was 840 bp long, the same as the coding sequence of HGF-beta. The cDNA of the shorter PT-PCR product was cloned, and the nucleotide sequence was found to concur precisely with the published mouse HGF-beta cDNA sequence (See SEQ ID NO: 1 1). Furthermore, although the HGF-alpha chain cDNA was completely absent, the signal sequence was identical to that in full-length HGF cDNA.

[00260] EXAMPLE 9

[00261] Formation and Biological Activity of Heterodimes of rIL-7 (SEQ ID NO:7, 8) and rHGF-beta (SEQ ID NO:3, 4, 13, and 14), Production of Recombinant HGF-beta Proteins. The PCR-amplified splice variant was subcloned into the mammalian expression vector pcDNA3.1 (+) (Invitrogen). The plasmid was transfected into Chinese hamster ovary (CHO) cells (LIPOFECTAMINE Plus™ Reagent, Life Technologies). The serum-free supernatant from the transfected CHO cells was collected, concentrated 10 times and filtered to remove any cells and evaluated for the production of HGF-beta protein (SEQ ID NO:3, 4, 13, and 14) by ELISA using anti-HGF-beta antibodies. The supernatant of transfected cells with vector but without HGF-beta gene was used as control medium. HGF-beta protein was detected in the supernatant of HGF-beta-transfected cells, but not in that of the empty vector-transfected cells (FIG. 14). The HGF-beta gene (See SEQ ID NO:9) was also subcloned into prokaryotic fusion protein expression vector pCAL-n (Stratagene, La Jolla, Calif.) and transformed into E.coli BL21(DE3). The fusion protein was purified by calmodulin affinity, and rHGF-beta, released by thrombin, was detected as a single band by SDS-PAGE and Western blotting.

[00262] EXAMPLE 10

[00263] Formation of a Biological Activive of Heterodimer of rIL-7 (SEQ ID NO: 7, 8) and rHGF-beta (SEQ ID NO:3, 4, 13, and 14) Using Low Molecular Weight Heparin Sulfate Oligosaccharides. As both IL-7 (SEQ ID NO:7, 8) and HGF (SEQ ID NO:5, 6) are heparin-binding molecules, the present inventors tested the ability of rIL-7 and rHGF-beta (SEQ ID NO:3, 4, 13, and 14) to from a heterodimer when equimolar ratios were mixed in serum-free medium in the presence or absence of low molecular weight heparin sulfate (HS)-derived oligosaccharides. The reactants were electrophoresed under nonreducing conditions and subjected to Western blot analysis. The results in FIG. 17 show that rHGF-beta migrated at 30 kD when added to rIL-7 in the absence of the HS-derived oligosaccharides, and at 45 kD in their presence. Similarly, rIL-7 migrated at 14.5 kD when added to rHGF-beta in absence of HS- derived oligosaccharides, and at 45 kD in their presence. Hence, rIL-7 and HGF-beta form an heterodimer in the presence of low molecular weight HS-derived oligosaccharides. Comparable results were obtained when FBS, rather than HS-derived oligosaccharides, was added to the medium.

[00264] To determine if the IL-7/HGF-beta heterodimer had biological activity, freshly harvested rat BM cells were incubated in culture medium containing 20% FBS plus rIL-7 and/or 2 times supernatant of HGF-beta transfected cells in the absence of anti-HGF-beta Ab. Lymphoid cells were harvested and phenotyped on day 10. Both rIL-7 and the supernatant of HGF-beta-gene transfected cells (or purified rHGF.beta. therefrom) were able to maintain the viability of pre-pro-B cells, but neither was able to stimulate their proliferation or to induce their differentiation to pro-B cells. However, when added concurrently, these reagents stimulated a significant increase in the generation of both pre-pro-B cells and pro-B cells (FIG. 15). Furthermore, this activity could be neutralized by anti-HGF-beta antibody. Similar results were obtained when the purified heterodimer of rIL-7 and rHGF-beta performed in the presence of HS-derived oligosaccharides, was used.

[00265] EXAMPLE 11

[00266] Analysis of B-Cell Development in Interleukin (IL)-7 Gene-deleted Mice, Maturation Arrest Occurs at the Late pro-B Cell Stage. Bone marrow cells from IL-7 K/O mice were stained for the expression of B220, CD43, HSA and/or BP-1 , as analyzed by FCM according to the scheme of Hardy et al, J. Exp. Med. 173 : 1213-1225 (1991). The data in FIG. 18 indicate that: (a) the number of Fr A (pre-pro-B) cells is normal; (b) Fr B and C (pro-B) cells are slightly reduced; and (c) Fr C, which is the transitional stage from pro-B to pre-B cells is missing. Furthermore, the expression of CD25, which has been suggested to distinguish cells undergoing VDJ from those undergoing DJ gene rearrangement was severely reduced. In addition, up-regulation of IL-7R-alpha and TdT, normally observed during pre-pro-B to pro-B cell differentiation, did not occur (FIGS. 19 and 20); neither did ομ expression of the late pro-B cell (transitional) stage (FIG. 21). Hence, although IL-7 (SEQ ID NO:7, 8) is not essential for the development of pro-B cells in BM, it is necessary for several key aspects of their differentiation.

[00267] EXAMPLE 12

[00268] Similar Defects Occur in yc K/O and Jak3 K/O Mice. Like their counterparts in IL-7 K/O mice, pro-B cells from gamma-c K/O and Jak3 K/O mice arrest at Fr C and express abnormally low levels of TdT, IL-7R-alpha and ομ. Conversely, despite also arresting at Fr C, pro-B cells from lambda5 K/O mice and RAG-1 K/O mice upregulate TdT and IL-7R- alpha, and the former express normal levels of ομ. Taken together, these results suggest that signaling through the IL-7R-alpha/gamma-c/Jak3 complex is essential for upregulation of TdT and IL-7R- alpha, for expression of ομ, and for the efficient development of cells beyond the pro-B cell stage.

[00269] EXAMPLE 13

[00270] Effect of PPBSF and rIL-7 in IL-7 K/O Mice. Because rIL-7 (SEQ ID NO:7, 8) supported the proliferation and differentiation of pro-B cells from IL-7 K/O mice in the presence of IL-7(-/-) BM stromal cells, and IL-7-depleted CM from IL-7 +/+ stromal cells was not functionally reconstituted by rIL-7, it was suspected that PPBSF, the heterodimeric form of IL-7, was the responsible factor in the pro-B culture system. This was confirmed by demonstrating that pretreatment with purified PPBSF enabled pro-B cells from IL-7 K/O mice to respond to rIL-7. The in vivo administration of rIL-7 to IL-7 K/O mice may correct the abnormalities in pro-B cell development by forming PPBSF in BM with the stromal cell derived PPBSF cofactor. It may be postulated that the in vivo administration of anti-IL-7 mAb prevents the production or survival of pro-B cells in IL-7 competent mice by simultaneously eliminating monomeric IL-7 and PPBSF. However, some level of redundancy by other cytokines, such as IL-3 and TSLP, may partially compensate for the role of PPBSF. The fact that both components of the PPBSF heterodimer are avidly bound by heparin sulfate oligosaccharides, as disclosed, may suggest that PPBSF may function as a cell surface and/or ECM-bound molecular complex.

[00271] A more specific answer to the above question appears to reside in the ability of PPBSF to selectively regulate the proliferation and differentiation of pre-pro-B cells, which normally express low levels of the IL-7R-alpha; and of monomeric IL-7 to regulate the Gl/S transition and differentiation of pro-B cells, which normally express high levels of the IL- 7R.alpha.. As PPBSF does not stimulate proliferation of pro-B cells and IL-7 does not stimulate proliferation of pre-pro-B cells, it would appear that PPBSF induces pre-pro-B cells to become IL-7-responsive pro-B cells by up-regulating the expression of IL-7R.alpha.

[00272] EXAMPLE 14

[00273] PPBSF, but not rIL-7 (SEQ ID NO:7, 8), Upregulates TdT and IL-7R-alpha on pro-B Cells From IL-K/O Mice In Vitro. rIL-7 (SEQ I DNO:7, 8) stimulated marked in vitro proliferation of pro-B cells from IL-7(+/+) and RAG-1(-/-) mice, but not from IL-7(-/-) mice. Conversely, native PPBSF (but not rIL-7 or PPBSF-coF alone) not only upregulated the expression of IL-7R-alpha, TdT, and ομ in pro-B cells from IL-7(-/-) mice, but "primed" them to proliferate in response to rIL-7. These results strongly support our working hypothesis that, in addition to stimulating the proliferation and differentiation of pre-pro-B cells, PPBSF upregulates the expression of high affinity IL-7R, thereby enabling pro-B cells to respond to monomeric IL-7. Significantly, PPBSF also "primed" pro-B cells from IL-7 K/O mice to proliferate to rIL-3 in the absence of IL-7.

[00274] EXAMPLE 15

[00275] In Vivo Treatment with rIL-7 (SEQ ID NO:7, 8) Restores B Cell Development in IL-7 K/O Mice. IL-7 K/O mice were injected i.p. daily with rIL-7 to confirm the essential role of IL-7 on early B cell development in vivo. As shown in FIG. 22A, the proportion of B220+CD43+ (Fr. A-C) cells among total BM cells progressively increased during the first 7 days of treatment, and that of B220+ CD43- (Fr. D-F) cells between days 7 and 12. Subset analysis with HSA (FIG. 22B) and slgM (FIG. 22C) showed sequentially overlapping increases in pro-B cells (Fr. B-C), transitional cells (Fr. C), pre-B cells (Fr. D) and immature B cells (Fr. E) between days 4 and 12. However, at day 12, the proportion of mature B cells (Fr. F) did not exceed that at day 0 (i.e. 10% of normal).

[00276] Other studies have shown that within 4 days of i.p. administration of rIL-7 (SEQ ID NO:7, 8), the expression of IL-7R-alpha, TdT and ομ. by pro-B cells and transitional pre-B cells was restored to normal levels in BM of IL-7 K/O mice; and these cells were responsive to further stimulation with rIL-7 in vitro. Pre-B cell development was detected in vivo by day 7 of rIL-7 treatment, and sIgM+ B-cell development by day nine. It remains to be determined if the effects of IL-7 reconstitution in IL-7 K/O mice (especially at the pre-pro-B and pro-B cell stages) is due to the formation of PPBSF in vivo.

[00277] EXAMPLE 16

[00278] In Vivo Treatment with rIL-7 (SEQ ID NO:7, 8) enables B220+CD43+ B- lineage Cells From IL-7 K/O Mice to Respond to rIL-7 In Vitro. The present inventors have previously demonstrated that pro-B cells in IL-7 K/O mice express abnormally low levels of IL- 7R-alpha, TdT and ομ, and that expression of these proteins increases to normal levels after in vivo reconstitution with rIL-7 (SEQ ID NO:7, 8).

[00279] As illustrated in FIG. 23 A that BM cells from IL-7 K/O mice, unlike those from WT and RAG-1 K/O mice, fail to generate B220+CD43+ pre-pro-B/pro-B cells in vitro when stimulated with graded concentrations of rIL-7 (SEQ ID NO:7, 8). In contrast, BM cells from day 4 rIL-7-reconstituted IL-7 K/O mice were as efficient as were those from IL-7 competent mice in generating B220+CD43+ cells when stimulated in vitro with rIL-7 (FIG. 23B). The RAG-1 K/O mice, whose pro-B cells express normal levels of IL-7R-alpha and TdT, were included to control for the absence of c, expression, pre-BCR formation and Fr C cells in IL-7 K/O mice. Although these defects substantially reduced the maximal level of responsiveness to rIL-7 (FIG. 23 A), a 20-fold increase in the number of B220+ CD43+ cell/well above input levels was still observed.

[00280] EXAMPLE 17 [00281] Both rIL-7 (SEQ ID NO:7, 8) and BM Stromal Cell-derived Signals are Required to Stimulate B220+CD43+ Cells from IL-7 K O Mice In Vitro. Since rIL-7 (SEQ ID NO:7, 8) was not sufficient to stimulate B220+CD43+ BM cells from untreated IL-7 K/O mice to proliferate in vitro, it was determined whether BM stromal cell layers could provide the necessary additional signals. As shown in FIG. 24, IL-7(-/-) as well as IL-7(+/+) BM stromal cell layers enabled rIL-7 to support the growth of B-lineage cells in vitro. Although, IL-7(+/+) BM stromal cells were sufficient by themselves, additional growth occurred in the presence of rIL-7.

[00282] Phenotypic analysis of the IL-7 K/O B-lineage cells generated in primary cultures containing IL-7-competent stromal cells showed that approximately 40% had matured to at least the pre-B cell stage (B220+CD43-) and that additional rIL-7 was not required, (FIG. 25A). Examination of the pre-pro-B/pro-B (B220+CD43+) cell fraction from these cultures (FIG. 25B) revealed that the expression of IL-7R-alpha was up-regulated on about 45% of the IL-7 K/O cells. Furthermore, when the B220+ cells from these primary cultures were transferred to stromal cell-deficient secondary cultures, they continued to proliferate in response to rIL-7 alone (FIG. 26).

[00283] EXAMPLE 18

[00284] Up-regulation of IL-7R-alpha Expression on B220+CD43+ Cells From IL-7 K/O Mice In Vitro. The growth to the pre-pro-B and pro-B cell stages was restricted by using the stromal-cell-dependent pro-B cell culture system, as optimized for mouse BM cells by the presence of both IL-7(+/+) BM stromal cells and 10 μg/ml rIL-7. As in the pre-B-type cultures (FIG. 25B), up-regulated expression of IL-7R-alpha was observed on approximately 50% of the IL-7 K/O B220+CD43+ cells generated in pro-B-type cultures.

[00285] Similar results were obtained when CM was substituted for the IL-7(+/+) stromal cell layers in these cultures (FIG. 27). However, although supplementation with rIL-7 was still required to amplify cell growth, rIL-7 was not able to restore lymphostimulatory activity to CM that had been adsorbed with anti-IL-7 mAb. This observation suggests that the active factor in CM was the heterodimeric form of IL-7, i.e. PPBSF. This was confirmed by demonstrating that purified PPBSF plus rIL-7 (SEQ ID NO: 7, 8) could stimulate the in vitro generation of B220+CD43+ cells (FIG. 28). Furthermore, as shown in FIG. 29, PPBSF and rIL-7 appeared to act sequentially. Thus, when BM cells from IL-7 K/O mice were cultured in primary cultures containing PPBSF only, the surviving cells were able to generate B220+CD43+ cells when transferred to secondary cultures containing rIL-7 only. In contrast, although initial exposure to rIL-7 alone maintained the viability of the B220+CD43+ cells from IL-7 K/O BM, it did not enable these cells to proliferate when restimulated with rIL-7.

[00286] EXAMPLE 19

[00287] Expression and Purification of a Single-chain IL-7 (SEQ ID NO:7, 8)/HGF- beta (SEQ ID NO:3, 4, 13, and 14) Protein. Because the supernatants from the expressed insect cells had the highest thymocyte stimulatory activities (data not shown), this system was chosen for purification and biologic studies of the scIL-7/HGF-beta protein. Inasmuch as the theoretical isoelectricpoint of the scIL-7/HGF-beta is approximately 8, it was reasoned that the protein should carry a positive charge at pH 7, thereby allowing it to flow through DEAE and to bind to CM sepharose resins. However, scIL-7/HGF-beta proteins were eluted from both the DEAE and CM columns. Of importance, the protein that was eluted from the CM column was biologically active. This form of scIL-7/HGF-beta was detected by the goat anti-IL-7 antibody from R&D Systems, but not by the rabbit anti-IL-7 antibody. In contrast, the scIL-7/HGF-beta protein that was eluted from the DEAE column was biologically inactive and was detected by the rabbit, but not the goat, anti-IL-7. Furthermore, only the inactive form of IL-7/HGF-beta reacted with the rabbit anti-HGF-beta antibody, whereas both the active and inactive forms reacted with the goat anti-HGF-beta. In addition, the active form of scIL-7/HGF-beta had a molecular mass of 50 to 55 kDa, compared with 45 to 50 kDa for the inactive form. Inasmuch as the inactive form neither inhibited nor synergized with the active form of scIL-7/HGF-beta (data not shown), only the properties of the active form are described.

[00288] EXAMPLE 20

[00289] Effect of scIL-7 (SEQ ID NO:7, 8)/HGF-beta (SEQ ID NO:3, 4, 13, and 14) on Early B-lineage Development In Vitro. The activity of the scIL-7/HGF-beta protein on early B lymphocyte development was studied using freshly harvested rat and mouse BM cells. Consistent with previous results, primary rat BM cell cultures generated pre-pro-B cells and pro- B cells in the presence of scIL-7/HGF-beta, whereas cultures containing rIL-7 generated pro-B and pre-B cells almost exclusively (Figure 31).

[00290] As shown in Figure 31, mouse BM cells responded somewhat differently than did rat BM cells. Rather than containing B220⁺ cells only, approximately 60% of the mouse lymphoid cells in these cultures were B220^". Moreover, approximately two thirds of the B220^" cells had a CD43⁺ HSA^" phenotype, suggesting that some might be CLPs. To substantiate this, the protocol of Hardy and colleagues was used to demonstrate that approximately 0% of the total cells in the scIL-7/HGF-beta treated cultures had the AA4.1⁺ B220^"HSA^" CD4^low phenotype characteristic of CLPs (Fraction Ao), 15% were early pre-pro-B cells (Fr. Al), 5% were late pre-pro-B cells (Fr. A2), and 20% were pro-B cells (Fr. B-Cl). In contrast, Fr. Ao and Al cells were absent from cultures containing rIL-7, and only 0.5% were A2 cells. Instead, 65% were pro-B cells (Fr. B-C), and 10% were pre-B cells (Fr. D). Cultures containing equimolar amounts both of IL-7 and scIL-7/HGF-beta contained Fr. Ao through Fr. D cells. When converted into mean numbers of cells per well (Table 1), the results showed an average overall 16-fold increase in output over input numbers of early B-lineage precursors in the scIL-7/ HGF-beta cultures, and a 27-fold increase in the rIL-7 cultures. However, the increases seen in the scIL-7/HGF-beta cultures were restricted to the CLP (31 -fold), pre-pro-B (8-fold), and pro-B (10-fold) cell fractions, whereas those in the IL-7 cultures involved the pro-B (51 -fold) and the pre-B (32-fold) cell fractions only. Furthermore, the results in Figure 32A and 32B show that scIL-7/ HGF-beta selectively stimulated the proliferation of CLPs and pre-pro-B cells, and rIL-7 of pro-B cells and pre-B cells. Hence, the marked expansion of pro-B cells in the scIL-7/HGF-beta cultures appears to be due primarily to their differentiation from proliferating pro-B cells.

[00291] Table 1. Generation of B-Lineage Cells in IL-7⁷ Bone Marrow Cell Cultures Number of Cells/Well (xlO ⁴).

sclL-7/HGFB rIL-7

Cultured Pre- Pre- BM Cell CLP Pro-B Pro-B Pre-B Total CLP pro-B Pro-B Pre-B Total

Input

2 5 4 <1 9 2 5 4 <1 9

(Day 1)

Output

62 41 41 <1 144 0 2 205 32 239

(Day 17)

Fold

31 8 10 0 16 0 0 51 >32 27

Increase

[00292] EXAMPLE 21 [00293] Effect of scIL-7 (SEQ ID NO:7, 8)/HGF-beta (SEQ ID NO:3, 4, 13, and 14) on CFU-Si₂. To determine if the scIL-7/HGF-beta supported the survival or proliferation of more primitive lymphohemopoietic precursors than CLPs, irradiated mice were injected intravenously with culture-generated cells, and the number of macroscopically visible spleen colonies was determined 12 days later. Cells (lxl O⁶) from the scIL-7/HGF-beta cultures generated 30 ± 4.2 CFU-S₁₂ above background (minimal estimate due to partial confluency), as compared with a mean of only 1 colony for the IL-7 cultures. Histologic examination of the colonies generated by the scIL-7/HGF-beta-cultured cells showed that many contained a mixture of erythroid, myeloid, and megakaryocytic elements. Furthermore, comparison with the CFU-S]₂ activity in freshly harvested BM showed that, on a per-cell basis, the scIL-7/HGF-beta- stimulated cultures contained a normal frequency of CFU-S₁₂. However, as the total number of cells in these cultures approximated that in the original inoculum (2xl 0⁶), it was not possible to determine whether scIL-7/HGF-beta maintained prolonged survival (15 days) of CFU-Si₂ or stimulated their (or their precursors') proliferation. Therefore, experiments were repeated using cultures to which c-kitL (50 ng/mL) and flt-3L (50 ng/mL) had been added. Within 7 days, both the scIL/HGF-beta and rIL-7 cultures contained approximately 5 -fold more cells (8.8xl 0⁶ to 12.8xl 0⁶) than were present in the original inoculum. Again, only the scIL-7/HGF-beta cultures contained significant CFU-Si₂ activity above background (c-kitL plus flt-3L only). However, as the total CFU-S]₂ activity per culture well exceeded the input activity by approximately 5-fold, it would appear the number of CFU-Sj₂ in the scIL-7/HGF-beta cultures had expanded by at least that amount.

[00294] EXAMPLE 22

[00295] Effect of scIL-7 (SEQ ID NO:7, 8)/HGF-beta (SEQ ID NO:3, 4, 13, and 14) on Early B-lineage Development In Vivo. To determine whether the culture-generated cells had functional lymphoid progenitor activity in vivo, lxl 0⁶ cells from cultures containing equimolar amounts of rIL-7 or scIL-7/HGF-beta were injected intravenously into sublethally irradiated CD45-congenic mice. The results in Tables 1 and 2 show that, at 3 weeks, the cells from the scIL-7/HGF-beta cultures generated 3- to 4-fold more splenic B-lineage cells than did those from the rIL-7 cultures, and the proportion of donor-origin B-lineage cells that had passed the pro-B cell stage greatly exceeded that in recipients of IL-7-cultured cells (P < .01). Furthermore, the B-cell generative activity of lxl 0⁶ cells from the scIL-7/HGF -beta-containing cultures was equivalent quantitatively and qualitatively to that of a saturating dose (20x10⁶) of normal BM cells, even though the latter contains a heterogeneity of cell types that might serve as lymphoid progenitors.

Table. 2. In Vivo Lymphoid Progenitor Activity of Culture-Generated BM

[00297] Table. 3. Subsets of Donor-Origin B Cells in BM.

% of B-lineaqe cells

In Vitro Pre-pro-B Pre-B and

treatment and pro-B immature B rIL-7 85.3+11.8 14.4+11.3 sclL-7/HGFp *62.5+16.7 ^*36.8+17.8

Normal BM cells 61.4+5.2 38.5+5.2

*p< 05 between rIL-7 and sclL-7/HGFp-treated cells.

[00298] EXAMPLE 23

[00299] Identity of the Receptor Complex for scIL-7 (SEQ ID NO:7, 8)/HGF-beta (SEQ ID NO:3, 4, 13, and 14). Ordinarily, IL-7 binds to the alpha and gamma-c chains of the IL-7R whereas the alpha chain of mature HGF binds to the HGFR, c-Met. However, it is not known if the HGF-beta chain also binds to c-Met. Therefore, to gain some insight into the nature of the receptor(s) for the scIL-7/HGF-beta, antibodies specific for the IL-7R-alpha chain, the gamma-c chain or c-Met were added to cultures of mouse early B-lineage cells generated in the presence of rIL-7 or scIL-7/HGF-beta. As shown in Figure 33, the ability of scIL-7/HGF-beta to stimulate the proliferation of BM lymphoid cells was partially, but significantly (P < .05), inhibited by antibodies to c-Met as well as those to the IL-7R-alpha and gamma-c chains. Also, a mixture of anti-c-Met and anti-IL-7R-alpha antibodies showed greater inhibition than did either antibody alone. In contrast, only the antibodies to the IL-7R-alpha and gamma-c chains inhibited proliferation stimulated by rIL-7.

[00300] Consistent with these blocking experiments, approximately 60% of the scIL- 7/HGF-beta culture-generated CLPs and pre-pro-B cells expressed both c-Met and the IL-7R, whereas more than 80% at the rIL-7 culture-generated pro-B cells expressed the IL-7R only (data not shown). Furthermore, confocal microscopy revealed that the IL-7 and c-Met receptors existed as aggregates that had undergone patching and capping on cells stimulated with scIL-7/ HGF-beta but not rIL-7 (data not shown). Of interest, similar IL-7R/c-Met complexes were observed on enriched pre-pro-B cell fractions from normal (noncultured) BM, suggesting that they may have been stimulated by endogenous IL-7/HGF-beta. In addition, cross stimulation studies (Figure 32C-D) revealed that rIL-7 did not stimulate the proliferation of CLPs or pre- pro-B cells from scIL-7/HGF-beta cultures, and that scIL-7/HGF-beta did not stimulate pro-B cells or pre-B cells from rIL-7 cultures. However, as noted previously for native IL-7/HGF-beta, scIL-7/HGF-beta was able to "prime" pre-pro-B/pro-B cells to respond to rIL-7 (Figure 32D), presumably by up-regulating the IL-7R-alpha chain. Hence, in their aggregate, these results suggest that the scIL-7/HGF-beta hybrid cytokine binds coordinately to the IL-7 and HGF receptors on B-cell precursors, whereas rIL-7 binds to the IL-7R only. Direct evidence that both the IL-7R (alpha and gamma-c chains) and c-Met are major components of the receptor complex for IL-7/HGF-beta was provided on Western blots after purification of receptor proteins from culture-generated CLPs/pre-pro-B cells on a scIL-7/HGF-beta affinity gel (Figure 34). In addition, demonstration that the binding of scIL-7/HGF-beta to early B-lineage cells initiates signal transduction through both the IL-7R and c-Met was provided by analysis of phosphorylation of Jal<3 (which associates with the gamma-c chain of the IL-7R complex) and Western blotting with anti-c-Met phosphospecific antibody. Results in Figure 35 show that both rIL-7 and scIL-7/HGF-beta transduce signals through the IL-7R, but that only scIL-7/HGF-beta signals through c-Met as well.

[00301] EXAMPLE 24

[00302] rIL-7/HGFp treatment inhibits local tumor growth [00303] To determine whether rIL-7/HGFp has antitumor activity, murine CT-26 colon cancer cells were injected s.c. into syngeneic BALB/c mice and, at 2-day intervals from days 2- 24, the mice were injected at the tumor site with different doses of rIL-7/HGFp (2.5, 5, 15 and 30 μg/injection) or control vehicle (PBS). As shown in Figure 39 A, tumor growth was inhibited in a dose-responsive manner, with no detectable effect seen by day 30 at the 2.5μ level and >80% inhibition occurring at the 30μg level. To compare the antitumor effect of rIL-7/HGFp with its component cytokines, BALB/c mice were injected at the tumor site with optimal and equimolar amounts of rIL-7/HGFp (15 μg/injection), rIL-7 (5 μg/injection) and/or rHGFp (10 μg/injection), according to the above schedule. As shown in Figure 39B, rIL-7 alone or mixed with rHGFp inhibited local tumor growth by -30% by day 30, whereas rHGFp alone had no effect. In contrast, the rIL-7/HGFp hybrid cytokine inhibited tumor cell growth by -76% (pO.01). As shown in Figure 39C, the ability of rIL-7/HGFP to inhibit local tumor growth was not restricted to colon cancer, but applied to malignant melanoma as well.

[00304] To determine whether rIL-7/HGFp, rIL-7 and/or HGFp directly affect the growth of tumor cells, CT-26 colon and B16F10 melanoma cancer cells were cultured in vitro for 2 to 7 days in the presence of 20 to 150 ng/ml rIL-7/HGFp (or PBS), a dose range that has been shown to stimulate the proliferation of thymocytes and early B-lineage cells (Lai L., Zeff, RA, and Goldschneider I. A recombinant single-chain IL-7/HGFp hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-Si₂, CLPs, and pre-pro-B cells. Blood 2006;107:1776-8). The rate of tumor cell growth was not significantly different at any dose level of rIL-7/HGFp from those observed in control cultures or in cultures containing equimolar amounts of rIL-7 and/or HGFP (data not shown). Hence, the mechanism by which rIL-7/HGFp inhibits the growth of CT-26 and B 16F10 tumors in vivo would not appear to involve direct cytotoxic or cytostatic activities. This was confirmed by the results of experiments in nude mice (see below).

[00305] EXAMPLE 25

[00306] rIL-7/HGFp induces significant infiltration of DCs and T cells into the tumors

[00307] The presence of large proportions of TILs has been associated with favorable clinical outcomes in cancer patients (Andersson A, Yang SC, Huang M, et al. IL-7 promotes CXC 3 ligand-dependent T cell antitumor reactivity in lung cancer. J Immunol 2009;182:6951- 8; McBride WH, Thacker JD, Comora S, et al. Genetic modification of a murine fibrosarcoma to produce interleukin 7 stimulates host cell infiltration and tumor immunity. Cancer Res 1992; 52:3931-7; Li B, VanRoey MJ, Jooss K. Recombinant IL-7 enhances the potency of GM-CSF- secreting tumor cell immunotherapy. Clin Immunol 2007;123:155-65). We therefore determined whether rIL-7/HGFp, rIL-7 and/or rHGFp treatment enhanced the presence of TILs in the s.c. murine colon and melanoma tumors. To normalize for differences in tumor size at the time sacrifice, we assessed the numbers of TILs per mg tumor tissue. On day 30 after tumor inoculation, single-cell suspensions of tumor tissue from mice treated with equimolar amounts of rIL-7/HGF , rIL-7 and/or rHGF (see Figure 39B), were analyzed for CD4⁺ and CD8⁺ T cells by flow cytometry. Due to the role of DCs in the activation of T cells, we also analyzed for CDl lc⁺ tumor infiltrating DCs. As shown in Figure 40 A, rIL-7 alone or mixed with rHGF significantly, albeit modestly (<2-fold), increased the numbers of CD8⁺ and CD4⁺ T cells, but not DCs, in the tumors. Again, rHGF alone had no effect. In contrast, rIL-7/HGF increased the numbers of DCs as well as CD4⁺ and CD8⁺ T cells 4 to 6-fold above the levels in PBS- injected controls (p<0.01 between PBS and rIL-7/HGFP). Furthermore, as shown in Figure 40B, the DCs in the rIL-7/HGFp-treated tumors expressed significantly higher levels of CD80 and CD86 than did those in rIL-7 and/or rHGF -treated tumors, indicating that they had undergone activation and maturation. In addition to the tumors themselves, there was a parallel increase in the number of CD4⁺ and CD8⁺ T cells and activated DCs in the draining lymph node (DLNs) (Figure 41 ; and data not shown). Similar results were observed in rIL-7/HGF -treated B16F10 melanoma-bearing mice (data not shown).

[00308] EXAMPLE 26

[00309] rIL-7/HGFP-treatment induces a tumor-specific immunological response

[00310] The above results suggest that the mechanism by which rIL-7/HGF inhibits local tumor growth is immunological, involving TILs and activated DCs. To explore this possibility, we examined the number of IFN-y-producing cells from the DLNs and spleen of cytokine-treated tumor-bearing mice after in vitro stimulation with syngeneic homologous or heterologous tumor cells. Cultures containing single cell suspensions of DLNs or spleen cells (normalized to lxlO⁵ T cells/well) from day 30 CT-26 colon cancer-bearing mice that had been treated in vivo with equimolar amounts of rIL-7/HGFp, rIL-7 and/or rHGFp (or PBS) were stimulated with irradiated CT-26 cells. Cultures stimulated with irradiated 66.1 murine breast cancer cells served as specificity controls (Walser TC, Ma X, Kundu N, et al. Immune-mediated modulation of breast cancer growth and metastasis by the chemokine Mig (CXCL9) in a murine model. J Immunother 2007;30:490-498). After 2 days, the numbers of IFN-γ producing cells present were quantified by ELISPOT assays.

[00311] As shown in Figure 42A, the numbers of IFN-γ producing cells among the cultured DLN cells from rIL-7 and/or HGFp-treated tumors was 2 to 3 -fold higher than were those from PBS-treated controls after stimulation with CT-26 cells. As expected, treatment with rHGFp alone had no effect. In contrast, the number of IFN-γ producing cells among the cultured DLN cells from rIL-7/HGF -treated tumors was elevated 6 to 7-fold (p< 0.01). Identical results were obtained when splenocytes from these same animals were used (Figure 42B). However, there was no increase in IFN-γ producing cells in any of the cultures that were stimulated with breast cancer cells. Similarly, rIL-7/HGFp-treatment significantly increased the number of tumor-specific IFN-γ producing T cells in DLNs and spleens of B16F10 melanoma-bearing mice (data not shown). There results suggest rIL-7/HGFp-treatment greatly enhances both regional and systemic immunological responses to tumor-specific antigens in vivo.

[00312] EXAMPLE 27

[00313] rIL-7/HGFp inhibits the formation of pulmonary metastases in recipients of colon cancer and melanoma cells

[00314] To determine whether rIL-7/HGF could also inhibit metastatic disease, BALB /c mice were injected i.v. with CT-26 cancer cells to establish pulmonary metastases. The mice were then injected i.v. with 15 μg rIL-7/HGF or equimolar doses of rIL-7 and/or HGFp (or PBS) at 2-day intervals between days 2-18. The mice were euthanized on day 21, the lungs were removed and weighed, and tumor colonies on the surface of the lung were counted. As shown in Figure 43A, i.v. rIL-7/HGF treatment reduced the numbers of metastatic nodules on the lungs by approximately 5.5-fold, as compared with 1.3-fold after rIL-7 treatment. Proportionate decreases in lung weights and in the sizes of the metastatic nodules that did form also were observed (data not shown). Similar antimetastatic activity was observed in the lungs of rlL- 7/HGF -treated C57BL6 mice after i.v. injection of melanoma cells (Figure 43B). Of interest, randomly sampled peripheral LNs contained significantly elevated numbers of CD4⁺ and CD8⁺ T cells and activated DCs following the systemic administration of rIL-7/HGFp similar to those observed in the DLNs after the local administration of rIL-7/HGFp (data not shown). These results suggest that DCs and T cells also may be involved in the antimetastatic activity of rlL- 7/HGFp.

[00315] EXAMPLE 28

[00316] Inhibition of local tumor growth by rIL-7/HGFP does not occur in nude mice.

[00317] To confirm that T cells were involved in the antitumor activity of rIL-7/HGFp, the previous experiments were repeated in congenitally athymic (nude) mice. In contrast to its effects in euthymic mice, rIL-7/HGFp failed to inhibit local tumor growth in nude mice (Figure 44A, B) by both CT-26 colon cancer and B16F10 melanoma cells. In contrast, the antimetastatic activity of rIL-7/HGF was only partly lost in nude mice (Figure 44C, D). These results suggested that, even if the expanded T cells and activated DCs seen in rIL-7/HGFp-treated euthymic mice inhibit the formation of metastases (Figure 43), rIL-7/HGFp must use a different mechanism(s) to achieve the same result in nude mice.

[00318] EXAMPLE 29

[00319] The ability of IL-7/HGFp hybrid cytokine to inhibit local tumor growth, in vivo, is a generalized effect.

[00320] Figure 45 demonstrates that, similar to the in vivo effect on colon and melanoma cancers, the IL-7/HGFp hybrid cytokine is also able to inhibit local tumor growth in vivo of a broad variety of cancers. In Figure 45 P388D leukemia cells (2X10⁵) (A) or Line 66.1 breast cancer cells (3X10⁵) (B) were injected s.c. into DBA/2, and BALB/c mice, respectively. Beginning on the day of tumor challenge, groups of mice were injected with rIL-7/HGFp (15 ug/injection), or control vehicle (PBS) at 2-day intervals. Tumor diameters were measured twice weekly. Data are reported as mean tumor diameter + SD from 5 mice each group. In Figure 46 C57BL/6 (B6) mice were injected s.c. with 5 X 10⁶ syngeneic TRAMP-C1 prostate cancer cells. When the mice developed palpable tumors, groups of the mice were injected i.p. with rlL- 7/HGF (15 ug/injection), or control vehicle (PBS) at 2-day intervals. Tumor sizes were measured periodically. Data are reported as mean tumor diameter + SD from 5 mice each group.

[00321] These data, together with the colon and melanoma cancer cell data, demonstrate that the ability of the recombinant IL-7/HGF hybrid cytokine to inhibit local tumor growth in vivo is a generalized effect. In addition, Figure 46 indicates that the recombinant IL-7/HGFp hybrid cytokine might also facilitate reduction in the volume of an established tumor and that this can be induced by the systemic as well as the intratumoral injection of rIL-7/HGFb. As such, the therapeutic compositions provided by the invention are suitable for use in methods to treat many different types of cancers in addition to those described in the examples. Such methods are expressly contemplated and ecompassed by the invention.

[00322] Exemplary Materials and Methods.

[00323] Animals. 129XB6F2 IL-7(-/-) and IL-7(-/-) mice (generously provided by Dr Richard Murray, DNAX Research Institute of Cellular and Molecular Biology, Palo Alto, CA) and Lewis strain rats were used as donors and/or recipients of BM lymphoid precursor cells and thymocytes.

[00324] Murine CT-26 colon cancer and B16F10 melanoma cells were obtained from the National Cancer Institute (Frederick, MD) and the American Type Culture Collection. Murine breast cancer 66.1 cell line was kindly provided by Drs. A. M. Fulton and N. Kundu (University of Maryland). BALB/c, C57BL/6 and congenitally athymic nude mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and the National Cancer Institute (Frederick, MD). Mice were housed, treated, and handled in accordance with the guidelines set forth by the University of Connecticut Health Center Animal Care Committee. rIL-7 was purchased from PreproTech (Rocky Hill, NJ) and R&D systems (Minneapolis, MN). rHGFp and rIL-7/HGFp was cloned, expressed, and purified as we have described (Lai L. and Goldschneider I. Cutting Edge: Identification of a hybrid cytokine consisting of Interleukin-7 and the β-chain of the Heptocyte Growth Factor/Scatter Factor. J Immunol 2001 ; 167:3550-4; Lai L., Zeff, RA, and Goldschneider I. A recombinant single-chain IL-7/HGF hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-Si₂, CLPs, and pre-pro-B cells. Blood 2006;107: 1776-84).

[00325] Cytokines and antibodies. Recombinant IL-7 (SEQ ID NO:7, 8) and goat polyclonal antibodies (Abs) against mouse IL-7 (SEQ ID NO: 8), IL-7R-alpha, gamma-c chain, and c-Met (R&D Systems, Minneapolis, MN); goat and rabbit polyclonal antibodies against human and mouse HGF-beta, rabbit anti-IL-7, and horseradish peroxidase (HRP)-linked anti- goat and anti-mouse immunoglobulin G (IgG) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); HRP anti-rabbit IgG antibody (Amersham Biosciences, Piscataway, NJ); phycoerythrin (PE), biotin-conjugated, and unconjugated mouse monoclonal antibodies (mAbs) against rat HIS24 (CD45R-B220), HIS 17 (CD43), HIS50 (HSA), and fluorescein isothiocyanate (FITC), PE, biotin, and allophycocyanin (APC)-conjugated anti-mouse B220, anti-HSAand anti-CD43 Abs (BD Biosciences, San Diego, CA); FITC and APC anti-IL-7R-alpha, APC anti- mouse CD4, and PE anti-AA4.1 (Bioscience, San Diego, CA); Alexa fluor 594-labeled anti-goat IgG (Molecular Probes, Eugene, OR); anti-CD 19, B220, and PE MicroBeads (Miltenyi Biotec, Auburn, CA); anti-Jak3 and phosphotyrosine (4G10) (Upstate Biotechnology, Lake Placid, NY); and anti-phospho-c-Met (Biosource, Camarillo, CA).

[00326] Thymidine incorporation. Thymocytes or culture-generated BM lymphoid cells were seeded in triplicate into 96- well plates, and 0.074 MBq (2 μθϊ) [³H] thymidine was added to each well 12 hours before completion of the 72-hour incubation period. Incorporation of [³H] thymidine was determined by liquid scintillation spectroscopy.

[00327] BrdU incorporation. BrdU solution (10 μΕ of 1 mM; BD Biosciences) was added per milliliter of cultured BM cells. The treated cells were incubated at 37°C for 45 minutes, and stained with fluorescent antibodies for cell-surface markers. After fixation and treatment with DNase, the cells were stained with APC-labeled anti-BrdU antibody and analyzed by flow cytometry.

[00328] Evaluation of local tumor growth and pulmonary metastasis

[00329] Cancer cells in the exponential growth phase were harvested and washed in PBS before in vivo injection. To induce localized tumors, 2xl0⁵ CT-26 colon cancer cells (Corbett TH, Griswold DP Jr., Roberts BJ et al. Tumor induction relationships in development of transplantable cancers of the colon in mice for chemotherapy assays, with a note on carcinogen structure. Cancer Res 1975; 35: 2434-39) or lxlO⁵ B16F10 melanoma cells were injected subcutaneously (s.c.) into the flank of syngeneic BALB/c or C57BL/6 mice, respectively. The indicated doses of rIL-7/HGFp, rIL-7 and/or rHGF (or PBS) were then injected s.c. into the tumor injection site at 2-day intervals over the indicated time period. Tumor size (volume) was determined twice weekly by caliper measurements of the shortest (A) and longest (B) diameter, using the formula V=(A B)/2. To induce pulmonary metastases, 2x10 CT-26 or B16F10 cancer cells were injected into the tail vein of syngeneic mice, and rIL-7/HGF or PBS were injected intravenously (i.v.) at 2-day intervals from days 2-18. The animals were euthanized at the indicated times after tumor inoculation. Metastatic tumor nodules in the subpleural regions of the lungs were counted under a dissecting microscope. [00330] Evaluation of TILs and DCs

[00331] At the indicated time points, the s.c. tumors were excised, weighed, minced into small fragments, and digested in 1 mg/ml collagenase IV (Sigma, St. Louis, MO) and 0.1 mg/ml DNase (Sigma, St. Louis, MO) at 37°C for 1 hour. The dissociated cells were filtered through a 35-μιη filter and prepared for phenotypic analysis of T cells and DCs by flow immunocytometry.

[00332] Flow immunocytometry

[00333] Single-cell suspensions from tumors and draining or non-draining lymph nodes were stained with the following fluorochrome-conjugated antibodies: CD4, CD8, CD1 lc, CD80, CD86 (BioLegend or BD Biosciences, San Diego, CA). The samples were analyzed on a FACSCalibur flow cytometer (Becton and Dickinson) with CellQuest acquisition software. Data analysis was done using FlowJo software (Ashland, OR).

[00334] Immunomagnetic cell separation. BM cells were stained with CD 19 MicroBeads for 15 minutes at 4°C, washed, and then applied to a magnetic-activated cell sorter (MACS) magnetic column. The CD 19^" cells were reacted with anti-B220 MicroBeads, and the CD19^"B220⁺ cells were isolated by immunomagnetic separation (IMS). Alternately, CD 19^" cells were stained with PE-anti-AA4.1 Ab and anti-PE MicroBeads, and the CD 19^"AA4.1⁺ cells were collected.

[00335] Confocal microscopy. Cytospin preparations of culture-generated BM lymphoid cells or enriched pre-pro-B cells from fresh BM were fixed with 4% paraformaldehyde and stained with FITC-labeled rat anti-IL-7R-alpha Ab or FITC-labeled rat IgG2a (isotype control) and purified goat anti-c-Met or goat IgG (isotype control) developed with Alexa fluor 594- labeled anti-goat IgG. The cells were then observed under a Zeiss LSM510 Meta laser scanning confocal microscope equipped with a Plan-Apochromat 63x1.4 numeric aperture objective (Carl Zeiss, Thornwood, NY). Images were processed using Photoshop 7.0 (Adobe Systems, Mountain View, CA).

[00336] Western blotting. Samples were mixed with 2X SDS sample buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to Immobilon-P membranes (Millipore, Bedford, MA). After blocking with 5% blocking reagent, the membranes were incubated with primary antibodies, HRP-labeled secondary antibodies, and then developed with enhanced chemiluminescence (ECL; Amersham Biosciences). [00337] Construction and expression of a single-chain murine IL-7 (SEQ ID NO:8)/HGF-beta (SEQ ID NO:3, 4) protein in baculovirus-insect cell expression system.

The cDNA encoding murine IL-7 (SEQ ID NO: 12) was generated from cultured thymus stromal cells of IL-7(-/-) mice, and amplified with primers specifying the mature protein-coding region. Baculovirus transfer vector pAcGP67A (BD Biosciences) containing the gp67 secretion signal sequence was used for the expression and secretion of a scIL-7/HGF-beta protein in insect cells (Figure 30). In order to insert IL-7 into the vector and to construct IL-7 and HGF-beta (See SEQ ID NO: 11), DNA connected by a flexible linker encoding (Gly4Ser) IL-7 DNA was amplified with primers (Table 4) containing the 3' end of gp67 secretion signal sequence (primer A) and the linker sequence (primer B). The gp67 secretion signal sequence and a part of the polyhedrin promoter (containing the NgoMW site) were amplified from the vector with primers C and D. The polymerase chain reaction (PCR) products of IL-7 and the signal sequence (SS-IL-7) were combined and subjected to an additional round of PCR with primers C and B. Because the 5' end of the IL-7 PCR product overlaps the 3' end of the signal sequence, the IL-7 DNA was seamlessly fused to the signal sequence after overlap extension PCR. The cDNA encoding murine HGF-beta was amplified from plasmid DNA with primers containing the linker (primer E), and stop codon and Xbal site (primer F). SS-IL-7 and HGF-beta DNA were combined and subjected to an overlap extension PCR by primers C and F. As the linker sequences in the 3' end of SS-IL-7 and the 5' end of HGF-beta overlap, an SS-IL-7 linker IL-7/HGF-beta DNA was constructed. The construct was digested with NgoMW and Xbal, ligated into the NgoMW /Xbal sites of the transfer vector, and transformed into Escherichia coli DH5-alpha cells. The plamid DNA was purified and sequenced. Sf9 insect cells were cotransfected with the transfer vector and BaculoGold linearized baculovirus DNA (BD Biosciences) to construct a recombinant baculovirus containing the SS-IL-7 linker IL-7/HGF-beta DNA via homologous recombination. The recombinant baculovirus was plaque selected, and virus banks were generated according the instruction manual (BD Biosciences). Sf9 insect cells were then infected with the recombinant baculovirus to produce the scIL-7/HGF-beta protein. The highest protein expression level was achieved at a multiplicity of infection (MOI) of 2 in suspension culture (26°C for 96 hours) using SF900II serum-free medium (SFM; Invitrogen, Carlsbad, CA) without protease inhibitor. [00338] Table 4. Primers used for cloning of IL-7/HGFP into expression vectors.

Underlined

Primers Primer Sequence (5 '-3 ') sequences

A GCGCATTCTGCCTTTGCGGAGTGCCACATTAAAGAC NA

B CGACCCACCACCGCCCGAGCCACCGCCTCCTATACTGCCCTTCAAAAT Linker

C GGGATCGTCGGTTTTGTA NA

D CGCAAAGGCAGAATGCGC NA

E GGAGGCGGTGGCTCGGGCGGTGGTGGGTCGGTTGTAAATGGCATTCCA Linker

F TGCTCTAGACTATTACAACTTGTATGTCAA Xba I site

G CGCGGCGCGCCGAGTGCCACATTAAAGAC Asc I site

H CGCCTCGAGCTATTACAACTTGTATGTCAA Xho I site

I CGCCTCGAGAAAAGAGAGTGCCACATTAAAGAC Xho I site

[00339] Expression of the single-chain IL-7 (SEQ ID NO:7, 8)/HGF-beta (SEQ ID NO:3, 4, 13, and 14) protein in mammalian and yeast expression systems. The IL-7-linker- HGF-beta (IL-7/HGF-beta) DNA (See SEQ ID NO:9-12) was also subcloned into mammalian expression vector pSecTag2A containing signal IgK sequence (Invitrogen) with AscllXhol sites (primers G, H). The plasmid DNA was transfected into Chinese hamster ovary (CHO) cells. To express and secrete the single-chain IL-7/HGF-beta protein in a yeast expression system, a yeast expression vector was modified by insertion of a Saccharomyces cerevisiae (SC) alpha-factor secretion signal (cut with Hindlll/Xbal) from pPIC6-alpha-A vector into Hindlll/Xbal sites of YES2 (Invitrogen). IL-7/HGF-beta DNA was subcloned into the modified vector with Xhol/Xbal sites (primers I and F; Table 4). The vector containing the IL-7/HGF-beta DNA was transformed into competent INVScl cells with S.c. EasyComp Transformtion Kit (Invitrogen) and the transformants were selected with SC-U selection plates. The INVScl cells were cultured in SC- U medium containing 2% glucose or raffinose and then induced by 2% galactose for another 4 to 22 hours.

[00340] Protein production and purification. The culture supernatant from the recombinant baculovirus infected Sf9 cells was concentrated by a Prep/scale-tangential flow filter (TFF) cartridge with 10 kDa molecular weight (MW) cut-off (Millipore, Bedford, MA) and diafiltered into washing buffer (30 mM Na₂P0₄, pH 7), The samples were then applied to serially linked columns of DEAE and CM sepharose (Amersham Biosciences). After washing, the linked columns were separated, and proteins were eluted from each by stepwise NaCl gradient from 25 mM to 300 mM in the washing buffer. The 200 to 300 mM CM eluates and the 37.5 to 100 mM DEAE eluates were pooled and loaded respectively on a Sephacryl S-200 column (Amersham Biosciences) pre-equilibrated with 30 mM Na₂P0₄ and 250 mM NaCl (pH 7). Fractions were collected and analyzed for IL-7/HGF-beta protein expression by Western blotting and thymocyte stimulating activity. Proteins were quantified by protein assay (Bio-Rad, Hercules, CA), using bovine serum albumin as a standard. Yields of up to 1 mg purified scIL-7/HGF-beta per liter of culture supernatant were obtained.

[00341] BM lymphoid cell culture and flow immunocytometric analysis. Rat and mouse femoral BM cells were collected by flushing with cold RPMI-1640, and the erythrocytes were lysed with 0.165 M NH₄C1. Nucleated cells (2xl0⁶) in 2 mL RPMI-1640 containing 5% lot selected, defined fetal bovine serum (FBS) and 5xl0^"5 M 2-mercaptoethanol (2-ME) were incubated in 35-mm diameter culture plate wells at 37°C in 5% C0₂ in the presence of rIL-7 (SEQ ID NO:7, 8) and/or scIL-7 (SEQ ID NO:7, 8)/HGF-beta (SEQ ID NO:3, 4, 13, and 14). Fourteen to 19 days later, the nonadherent cells were harvested for phenotypic analysis. The cell samples were analyzed by CELL Quest on a FACScan or FACScalibur flow cytometer (all from Becton Dickinson Immunocytometry Systems, San Jose, CA).

[00342] Immunoprecipitation of phospho-Jak3 and c-Met. Culture-generated B- lineage cells were washed, cytokine-starved for 5 hours, and stimulated with either rIL-7 (20 ng/mL) or rIL-7/HGF-beta (60 ng/mL) for 10 to 30 minutes. The stimulated and unstimulated control cells were lysed in 25 mM Tris-HCL (pH 8), 150 mM NaCl, 1% Triton, 0.5% Igepal, and 1 mM sodium ortho vanadate plus protease inhibitors. The supernatants were immunoprecipitated with antibodies against Jak3 or phosphotyrosine and protein G-agarose bead slurry. Pellets were resuspended in SDS sample buffer and subjected to Western blotting using antibodies against phosphotyrosine or phospho-c-Met.

[00343] Purification of the IL-7/HGF-beta receptor. scIL-7/HGF-beta (1 mg) was coupled to 0.2 mL cyanogen bromide (CNBr)-activated sepharose 4B according to the manufacturer's instructions. Purified CD 19^" AA4.1 culture-generated cells (10 ; CLPs and pre- pro-B cells) were added to 2 mL lysis buffer (10 mM Tris-HCl buffer [pH 7.2], 150 mM NaCl, and protease inhibitor cocktail in the presence of 1% Triton X-100), and the supernatant was added to a scIL-7/HGF-beta affinity gel and gently rocked overnight at 4°C as described. After extensive washing with the lysis buffer (10 mM Tris-HCl buffer [pH 7.2] containing 0.15 M NaCl, 0.1% Triton X-100, and the protease inhibitor cocktail), the IL-7/HGF-beta receptor was eluted in a stepwise fashion with 0.1 M glycine-HCL buffer (pH 3.3), 0.1 M glycine-HCl buffer (pH 2.0), and then 0.1 M sodium citrate buffer (pH 2.0) each containing 0.2 M NaCl, 0.1% Triton X-100, and the protease inhibitor cocktail. The eluates were immediately neutralized with 2 M Tris base and subjected to SDS-PAGE and Western blotting.

[00344] ELISPOT assay

[00345] ELISPOT assay measuring IFN-γ were used to assess the in vitro T cell responses to stimulation with CT-26 or B16F10 cells. Splenocytes, containing lxl 0⁵ T cells/well, and irradiated cancer cells (lxl0⁵/well) were incubated for 2 days in 96- well plates (Millipore) coated with anti-mouse IFN-γ antibody (clone R4-6A2, Biolegend) and blocked with RPMI media supplemented with 10%> fetal calf serum. The wells were then washed and incubated with biotinylated anti-mouse IFN-γ antibody (clone XMG1.2, Biolegend). Reactions were visualized and counted using the streptavidin-peroxidase system (Taguchi T, McGhee JR, Coffman RL, et al. Detection of individual mouse splenic T cells producing IFN-gamma and IL-5 using the enzyme-linked immunospot (ELISPOT) assay. J Immunol Methods 1990;128:65-73).

[00346] Statistical analysis

[00347] P-values were based on two-sided Student's t test. A confidence level above 95%> (p<0.05) was determined as significant.

[00348] Incorporation by Reference

[00349] The contents of all references, patents, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

[00350] Equivalents

[00351] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[00352] It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the invention. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and/or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present invention will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A method for inhibiting cancer cell growth, proliferation and/or metastasis, in vivo, comprising administering to an individual a composition comprising an effective amount of an IL-7/HGF- β hybrid cytokine, and a pharmaceutically acceptable carrier, wherein the composition is effective for inhibiting cancer cell growth, proliferation and/or metastasis, in vivo.

2. The method of claim 1, wherein the IL-7/HGF-P hybrid cytokine comprises a non-covalently bound complex of an IL-7 polypeptide or bioactive portion thereof, and an HGF-β polypeptide or bioactive portion thereof.

3. The method of claim 1, wherein the IL-7/HGF-P hybrid cytokine is a chimeric or fusion protein comprising an IL-7 polypeptide or bioactive portion thereof, and an HGF-β polypeptide or bioactive portion thereof, joined contiguously in a single polypeptide chain.

4. The method of claim 3, wherein the chimeric or fusion protein further comprises a linker peptide between the IL-7 polypeptide and the HGF-β polypeptide.

5. The method of claim 4, wherein the linker is from 1 to about 100 amino acids in length.

6. The method of claim 3, wherein the chimeric protein comprises a bioactive portion of HGF-β.

7. The method of claim 1 , wherein the cancer cell is a localized tumor cancer cell.

8. The method of claim 7, wherein the composition is administered subcutaneously.

9. The method of claim 1 , wherein the cancer cell is a metastatic cancer cell.

10. The method of claim 9, wherein the composition is administered intravenously.

1 1. The method of claim 1 , wherein the cancer cell is colon cancer cell.

12. The method of claim 1 , wherein the cancer cell is a melanoma cancer cell.

13. The method of claim 1 , wherein the cancer cell is a T cell lymphocytic cancer cell.

14. The method of claim 1 , wherein the cancer cell is a breast cancer cell.

15. The method of claim 1, wherein the cancer cell is a prostate cancer cell.

16. A method of increasing the number of tumor-infiltrating lymphocytes (TILs) and/or Dendritic Cells (DCs), in vivo, comprising administering to an individual having cancer a composition comprising an effective amount of an IL-7/HGF-P hybrid cytokine, wherein the composition is effective in increasing the number of TILs and/or DCs.

17. The method of claim 16, wherein the TILs are CD4+ and/or CD8+ T cell lymphocytes.

18. The method of claim 16, wherein the Dendritic Cells are CD80+ and/or CD86+ cells.

19. A method of creating a tumor-specific T cell, in vivo, comprising administering an effective amount of an IL-7/HGF-p hybrid cytokine to an individual suffering from cancer, wherein the IL-7/HGF- β hybrid cytokine is effective for enhancing the secretion of interferon-gamma (IFN-γ) by the T cell.

20. A method of inhibiting and/or treating pulmonary metastatic disease, in vivo, comprising administering a composition comprising an effective amount of an IL-7/HGF-P hybrid cytokine, and a pharmaceutically acceptable carrier to an individual having pulmonary metastatic disease, wherein the IL- 7/HGF-p hybrid cytokine is effective for inhibiting the growth and/or proliferation of pulmonary metastases.

21. The method of claim 20, wherein the IL-7/HGF-p hybrid cytokine is a chimeric or fusion protein comprising an IL-7 polypeptide and an HGF-β polypeptide joined contiguously in a single polypeptide chain.

22. The method of any of claims 1, 2, 3, or 19, wherein the IL-7 polypeptide has at least 70% sequence identity with SEQ ID NO: 7.

23. The method of any of claims 1, 2, 3, or 19, wherein the HGF-β polypeptide has at least 70% sequence identity with SEQ ID NO: 13 or 14.

24. A therapeutic composition comprising an effective amount of an IL-7/HGF-p hybrid cytokine in combination with an effective amount of at least one other anti-cancer agent, and a pharmaceutically acceptable carrier.

25. The therapeutic composition of claim 24, wherein the IL-7/HGF- hybrid cytokine is a chimeric or fusion protein comprising an IL-7 polypeptide or bioactive portion thereof, and an HGF-β polypeptide or bioactive portion thereof, joined contiguously in a single polypeptide chain; and optionally including a linker sequence therebetween.