[go: up one dir, main page]

WO2006105538A2 - Methodes et compositions de traitement de pathologies afferentes a l'il-21 - Google Patents

Methodes et compositions de traitement de pathologies afferentes a l'il-21 Download PDF

Info

Publication number
WO2006105538A2
WO2006105538A2 PCT/US2006/012749 US2006012749W WO2006105538A2 WO 2006105538 A2 WO2006105538 A2 WO 2006105538A2 US 2006012749 W US2006012749 W US 2006012749W WO 2006105538 A2 WO2006105538 A2 WO 2006105538A2
Authority
WO
WIPO (PCT)
Prior art keywords
agonist
mice
drug
human
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2006/012749
Other languages
English (en)
Other versions
WO2006105538A3 (fr
Inventor
Keying Ma
Xiao-Yu Song
Donald E. Grisworld
Xiaozhou Shang
Li Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Biotech Inc
Original Assignee
Centocor Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centocor Inc filed Critical Centocor Inc
Publication of WO2006105538A2 publication Critical patent/WO2006105538A2/fr
Anticipated expiration legal-status Critical
Publication of WO2006105538A3 publication Critical patent/WO2006105538A3/fr
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation

Definitions

  • the present invention relates to methods for treating at least one IL-21 related pathology, including therapeutic formulations, administration and devices.
  • Cytokines are important in haematopoiesis, lymphocyte development, and the initiation and maintenance of the immune response.
  • Interleukin 21 IL-21
  • IL-21 Interleukin 21
  • the human IL-21 gene resides in close proximity to the EL-2 gene on chromosome 4q that has been shown to have linkage susceptibility to inflammatory bowel disease (IBD) including Crohn's disease (CD) and ulcerative colitis.
  • IBD inflammatory bowel disease
  • CD Crohn's disease
  • ulcerative colitis the IL-21 receptor gene maps to 16pll, another genomic region associated with susceptibility to CD.
  • IL- 21 is secreted by activated T cells.
  • IL-21 receptor is expressed in lymphoid tissues, in particular by NK, B, T cell and dendritic cells, macrophages and endothelial cells.
  • NK natural killer
  • B T cell
  • dendritic cells macrophages and endothelial cells.
  • IL-21 plays a role in the proliferation and maturation of natural killer (NK) cell populations, the proliferation of mature B cells co-stimulated with anti-CD40, and the proliferation of T cells co-stimulated with anti-CD3.
  • IL-21 limits NK cell responses and promotes antigen-specific T cell activation, which suggests that IL-21 promotes the transition from innate to adaptive immunity.
  • IL-21 may play a unique role in fine-tuning the immune response that involves B, T, and NK cells, depending on the type of stimulus and phenotype of immune cells.
  • Other studies indicate that IL-21 down-regulates IgE production from B cells through the inhibition of germ line C ⁇ transcription and thus suggest that IL-21 may be useful for treatment of IgE- dependent allergic diseases.
  • IL-21 also activates both innate and adaptive immunity to generate potent anti-tumor responses and may be a potential therapeutic for cancer. [5] Accordingly, there is a need to provide methods and compositions for treating at least one EL-21 related pathology, that overcome one more of these problems.
  • the present invention provides methods and composition for treating at least one IL-21 agonist related pathology using at least one isolated IL-21 agonist, as well as IL-21 agonist compositions, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants, and methods of making and using thereof, as described and enabled herein, in combination with what is known in the art, for use in treating at least one IL-21 pathologies.
  • the at least one agonist used in methods or compositions of the present invention can optionally comprise at least one IL-21 specific agonist protein, compound, ligand, antibody fusion protein, or agonist antibody, or fragment thereof, that agonizes at least one IL-21 biological activity, in vitro, in vivo, or in situ, such as, but not limited to, interacting with, e.g., binding to, an IL-21 polypeptide (e.g., a human IL-21 polypeptide); associating with signal transduction molecules, e.g., .gammax, jakl; stimulating phosphorylation and/or activation of stat proteins, e.g., stat 5 and/or stat3; or modulating, e.g., stimulating or decreasing, proliferation, differentiation, effector cell function, cytolytic activity, cytokine secretion, and/or survival of immune cells, e.g., T cells (CD8+, CD4+ T cells), NK cells, B cells, macrophages and megakary
  • Such IL-21 agonists can also comprise any portion of an IL-21 protein or known variant that is sufficient to agonize at least one IL-21 receptor or ligand that results in inducing or enhancing at least one IL-21 biological activity.
  • Such IL-21 proteins can include an IL-21 polypeptide or fragment that binds or associates with at least one IL-21 receptor or IL-21 ligand.
  • interleukin-21 can optionally refer to a cytokine showing significant sequence homology to IL-2, BL-4 and EL- 15 (Parrish-Novak et al. (2000) Nature 408:57-63) and have substantial IL-21 biological activity. Despite low sequence homology among interleukin cytokines, cytokines share a common fold into a "four-helix-bundle” structure that is representative of the family. It is expressed primarily in activated CD4+ T cells, and has been reported to have effects on NK, B and T cells (Parrish-Novak et al. (2000) supra; Kasaian, M. T. et al. (2002) supra).
  • EL-21 or "IL-21 polypeptide” refers to a protein (preferably of mammalian, e.g., murine or human origin) which is capable of interacting with, e.g., binding to, IL-21R (preferably of mammalian, e.g., murine or human IL-21) and having one of the following features: (i) an amino acid sequence of a naturally occurring mammalian IL-21 or a fragment thereof, e.g., an amino acid sequence shown as SEQ ID NOS :42 or 51, or an analog, such as, but not limited to, SEQ ID NOS:44-50 (human) or any biologically active fragment thereof; (ii) an amino acid sequence substantially homologous to, e.g., at least
  • hybridizes under stringent conditions describes conditions for hybridization and washing.
  • Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used.
  • a preferred, example of stringent hybridization conditions are hybridization in 6.times.sodium chloride/sodium citrate (SSC) at about 45 degrees C. C, followed by one or more washes in 0.2 times SSC, 0.1% SDS at 50 degrees C. C.
  • Another example of stringent hybridization conditions are hybridization in 6 times SSC at about 45 degrees C.
  • stringent hybridization conditions are hybridization in 6 times SSC at about 45 degrees C. C, followed by one or more washes in 0.2 times SSC, 0.1% SDS at 60 degrees C. C.
  • stringent hybridization conditions are hybridization in 6 times SSC at about 45 degrees C. C, followed by one or more washes in 0.2 times SSC, 0. 1% SDS at 65 degrees C. C.
  • Particularly preferred highly stringent conditions are 0.5M sodium phosphate, 7% SDS at 65 degrees C. C, followed by one or more washes at 0.2 times SSC, 1% SDS at 65 degrees C. C.
  • Such IL-21 polypeptides can include but or not limited to at least one portion of a known IL-21 sequence (e.g., but not limited to a portion of SEQ ID NO:42 or any known variant thereof, e.g., SEQ ID NO:44, or any fragment or other variant as known in the art.
  • IL-21 agonists can also comprise any portion of an IL-21 chemical or small molecule agonist or known variant that is sufficient to agonize at least one IL-21 receptor or ligand that results in inducing or enhancing at least one IL-21 biological activity.
  • Such IL-21 agonists can also comprise an agonist antibody fragment that binds and agonizes at least one IL-21 receptor (e.g., SEQ ID NO:43, or a fragment or known variant thereof) or IL-21 ligand, can optionally comprise at least one CDR (complementarity determining region) (e.g., CDRl, CDR2 or CDR3 of at least one heavy and/or light chain variable region) specific and agonistic for at least one IL- 21 target or receptor and/or at least one framework region (e.g., FRl, FR2, FR3, FR4 or fragment thereof, included as part of an antibody, fragment or antibody or receptor fusion protein or molecule.
  • CDR complementarity determining region
  • framework region e.g., FRl, FR2, FR3, FR4 or fragment thereof, included as part of an antibody, fragment or antibody or receptor fusion protein or molecule.
  • the at least one agonist amino acid sequence can further optionally comprise at least one specified substitution, insertion or deletion as provided in Figures 1-41 of publication WO 05/33029 and US serial No. 10/872,932, filed 06/21/2004, entirely incorporated herein by reference, with corresponding SEQ ID NOS :31-72.
  • the at least one agonist can further optionally comprise at least one IL-21 receptor agonist binding sequence (e.g., a portion of an IL-21 polypeptide) and at least one portion of at least one heavy or light chain constant region, comprising at least one of 10-384 contiguous amino acids of at least one of SEQ ID NOS :31-41, or at least one CHl, hinge 1, hinge2, hinge 3, hinge4, CH2, or CH3 fragment thereof as described in Table 1, further optionally comprising at least one substitution, insertion or deletion as provided in Figures 1-41 of publication WO 05/33029 and US serial No. 10/872,932, filed 06/21/2004, entirely incorporated herein by reference.
  • at least one IL-21 receptor agonist binding sequence e.g., a portion of an IL-21 polypeptide
  • at least one heavy or light chain constant region comprising at least one of 10-384 contiguous amino acids of at least one of SEQ ID NOS :31-41, or at least one CHl, hinge 1, hinge2, hinge 3, hinge4,
  • the present invention also provides at least one agonist, comprising at least one target binding sequence and at least 10-384 contiguous amino acids of at least one of SEQ ID NOS: 1- 41, or at least one FRl, FR2, FR3, FR4, CHl, hingel, hinge2, hinge 3, hinge4, CH2, CH3 or fragment thereof as described in Table 1, further optionally comprising at least one substitution, insertion or deletion as provided in Figures 1-41 of publication WO 05/33029 and US serial No.
  • the present invention also provides at least one composition
  • a composition comprising (a) an isolated IL-21 agonist encoding nucleic acid and/or agonist as described herein; and (b) a suitable carrier or diluent.
  • the carrier or diluent can optionally be pharmaceutically acceptable, according to known methods.
  • the composition can optionally further comprise at least one further compound, protein or composition.
  • the present invention also provides at least one method for expressing at least one IL- 21 agonist in a host cell, comprising culturing a host cell as described herein and/or as known in the art under conditions wherein at least one 1L-21 agonist is expressed in detectable and/or recoverable amounts.
  • the present invention further provides at least one IL-21 agonist, specified portion or variant in a method or composition, when administered in a therapeutically effective amount, for modulation, for treating or reducing the symptoms of at least one IL-21 pathology, disease or condition in a cell, tissue, organ, animal or patient and/or, as needed in many different conditions, such as but not limited to, prior to, subsequent to, or during a related disease or treatment condition, as known in the art and/or as described herein.
  • the present invention also provides at least one composition, device and/or method of delivery of a therapeutically or prophylactically effective amount of at least one IL-21 agonist, according to the present invention.
  • the invention also provides at least one isolated IL-21 human agonist encoding nucleic acid, comprising a nucleic acid that hybridizes under stringent conditions, or has at least 95% identity, to a nucleic acid encoding an IL-21 agonist.
  • the invention further provides an isolated IL-21 human agonist, comprising an isolated human agonist encoded by such a nucleic acid.
  • the invention further provides an IL-21 human agonist encoding nucleic acid composition, comprising such an isolated nucleic acid and a carrier or diluent.
  • the invention further provides an agonist vector, comprising such a nucleic acid, wherein the vector optionally further comprises at least one promoter. Such a vector can optionally further comprise at least one selection gene or portion thereof.
  • the invention further comprises a mammalian host cell comprising such an isolated nucleic acid encoding at least one PROTNAME 1 protein or receptor agonist polypeptide.
  • the invention also provides at least one method for producing at least one IL-21 human agonist, comprising translating such a nucleic acid or an endogenous nucleic acid that hybridizes thereto under stringent conditions, under conditions in vitro, in vivo or in situ, such that the IL- 21 agonist is expressed in detectable or recoverable amounts.
  • the invention also provides at least one IL-21 human agonist composition, comprising at least one isolated IL-21 human agonist and a carrier or diluent, optionally further wherein said carrier or diluent is pharmaceutically acceptable, and/or optionally further comprising at least one compound or protein selected from at least one of a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid, a IL-21 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 erythropoi
  • the present invention also provides at least one method for treating a IL-21 condition in a cell, tissue, organ or animal, comprising contacting or administering a immune related- or infectious related-condition modulating effective amount of at least one IL-21 human agonist with, or to, said cell, tissue, organ or animal, optionally wherein said animal is a primate, optionally a monkey or a human.
  • the method can further optionally include wherein said effective amount is 0.001-100 mg/kilogram of said cells, tissue, organ or animal.
  • Such a method can further include wherein said contacting or said administrating is by at least one mode selected from intravenous, intramuscular, bolus, intraperitoneal, subcutaneous, respiratory, inhalation, nasal, vaginal, rectal, buccal, sublingual, intranasal, subdermal, or transdermal.
  • the present invention also provides at least one medical device, comprising at least one IL-21 human agonist, wherein said device is suitable to contacting or administering said at least one IL-21 human agonist by at least one mode selected from intravenous, intramuscular, bolus, intraperitoneal, subcutaneous, respiratory, inhalation, nasal, vaginal, rectal, buccal, sublingual, intranasal, subdermal, or transdermal.
  • the invention also includes at least one human IL-21 agonist; wherein said human agonist binds the same epitope or antigenic region as an IL-21 human agonist.
  • the invention further provides at least one method of treating at least one IL-21 mediated condition, comprising administering to a patient in need thereof a formulation of the invention.
  • the invention also provides at least one article of manufacture for human pharmaceutical use, comprising packaging material and a container comprising a solution or a lyophilized form of at least one IL-21 human agonist of the invention.
  • said container is a glass or plastic container having a stopper for multi-use administration, optionally further wherein said container is a blister pack, capable of being punctured and used in intravenous, intramuscular, bolus, intraperitoneal, subcutaneous, respiratory, inhalation, nasal, vaginal, rectal, buccal, sublingual, intranasal, subdermal, or transdermal administration;
  • said container is a component of a intravenous, intramuscular, bolus, intraperitoneal, subcutaneous, respiratory, inhalation, nasal, vaginal, rectal, buccal, sublingual, intranasal, subdermal, or transdermal delivery device or system;
  • said container is a component of an injector or pen-injector device or system for intravenous, intramuscular,
  • the invention also provides at least one method for producing at least one IL-21 human agonist of the invention, comprising providing a host cell, transgenic animal, transgenic plant or plant cell capable of expressing in recoverable amounts said human agonist, optionally further wherein said host cell is a mammalian cell, a plant cell or a yeast cell; said transgenic animal is a mammal; said transgenic mammal is selected from a goat, a cow, a sheep, a horse, a rodent, a mammal and a non-human primate.
  • the invention further provides at least one transgenic animal or plant expressing at least one human agonist of the invention.
  • the invention further provides at least one IL-21 human agonist produced by a method of the invention.
  • the invention further provides at least one method for treating at least one IL-21 mediated disorder, comprising at least one of (a) administering an effective amount of a composition or pharmaceutical composition comprising at least one IL-21 human agonist to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy; and further administering, before concurrently, and/or after said administering in (a) above, at least one selected from at least one of a immune related therapeutic, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid, a neurological agent, a mineral, a nutritional,
  • FIG. 1 is a pictorial description of wild-type allele (WT) and IL-2r A mice construct as shown on the top and bottom respectively.
  • the IL-21 gene-deficient construct consisting of a neomycin resistance cassette flanked by appropriate linkers for homologous recombination, was targeted to replace the IL-21 exon 1 and exon 2 sequences. '
  • Figures 2A and 2B are graphical and pictorical representation of data on genotyping IL-2Y' ⁇ ir ⁇ ce.
  • Figure 2(a) shows a Southern blot. Tail genomic DNA samples were digested with Apa I and probed with a 348 bp 3' internal probe which results in a ⁇ 28 kb wild type band and a ⁇ 20 kb targeted band. Alternatively, the tail genomic DNA samples were digested with Hind IH and probed with an 192 bp 5' external probe resulting in a 8 kb wild type band and a 5 kb targeted band.
  • Figure 2(b) show a typical genotyping result of Multiplex PCR.
  • FIG. 3 is a graphical representation showing that IL-21 7" mice have reduced number of CD8 T cells.
  • CD8 populations of splenocytes (top) and thymocytes (bottom) from WT (left) and TL-21 ' ' ' (right) littermates were analyzed for expression of CD8 and CD4 by fluorescence-activated cell sorting (FACS). The numbers shown in lower right represent the percentage of CD8 T cells. Shown are representative FACS profiles from ten mice in each group.
  • FIG. 4 is a graphical representation showing that IL-21-/- mice have reduced numbers of NK and NK T cells.
  • NKl.1 and CD3-NK1.1 populations of splenocytes (top), PBMC (bottom) from WT (left) and IL-21-/- (right) littermates were analyzed for expression of NK 1.1 and CD3 by FACS.
  • the numbers shown in lower right represent the percentage of NKl .1 cells, and upper right represent the percentage of CD3-NK1.1 cells. Shown are representative FACS profiles from ten mice in each group.
  • FIG. 5 is a graphical representation showing that IL-21-/- mice have increased numbers of granulocytes (GrI positive cells).
  • GrI populations in PBMC from WT (left) and IL-21-/- (right) littermates were analyzed for expression of CD4 and GrI by FACS. The numbers shown represent the percentage of cells with a granulocyte phenotype. Shown are representative FACS profiles from ten mice in each group.
  • Figure 6 is a graphical representation showing that spleen cells of IL-21 "7" mice showed a decreased response to anti-mCD3 and anti-mCD28 stimulation.
  • Spleen cells were cultured in a 96- well plate pre-coated with or without 10 Dg/ml of anti-murine CD3 monoclonal antibody (clone 145-2C11) or 5 Dg/ml of anti-murine CD28 and cell proliferation was determined 72 hours later. Each data point represents the mean ⁇ SEM of ten mice.
  • FIG. 7 Murine IL-21 inhibits JL- 15 mediated resting NK cell expansion.
  • Spleen cells from WT or JL-2V 1' mice were cultured for 7 days with 10 ng/ml of IL- 15 and 100 ng/ml of mIL-21.
  • NK cell subsets were identified as NKl .1 + /CD3 " by FACS. Shown are representative FACS profiles from ten mice in each group.
  • FIG. 10 Elevated IgE level in JL-21 " ' ' mice. Serum samples were collected from WT and JL-21 '1' mice. IgE levels were determined by ELISA. Each data point represents the mean ⁇ SEM often mice. Differences between WT and IL-21 ⁇ ' ⁇ mice were tested using student's t test (*p ⁇ 0.05).
  • Figure 10 Decreased ear swelling in IL-21"'" mice in oxazolone-induced acute and chronic CHS. oxazolone induced acute CHS response.
  • mice received a topical application of 1.6 % of oxazolone (W/V) onto both sides of the left ear on day 0 and were challenged with 0.8% of oxazolone onto both sides of the left ear on day 7. Each data point represents the mean of six mice.
  • (B) oxazolone induced chronic CHS response. Mice received a topical application of 1.6 % of oxazolone (W/V) onto both sides of the left ear on day 1 and then were challenged with 0.8% of oxazolone onto both sides of the left ear on days 8, 11, 15 and 18. Each data point represents the mean ⁇ SEM of eight mice. Differences between WT and IL-21 " ' " mice were tested using student's t test (*p ⁇ 0.05).
  • Figure 22 shows histologic appearance of oxazolone-induced contact sensitivity. Ear samples were obtained 48 hrs after oxazolone challenge and fixed in 10% neutral formalin.
  • Sections were stained with hematoxylin and eosin.
  • A vehicle-treated WT;
  • B oxazolone induced acute CHS response in WT;
  • C oxazolone induced acute CHS response in JL-21 "7" mice;
  • D oxazolone induced chronic CHS response in WT on day 19;
  • E oxazolone induced chronic CHS response in IL-21 ⁇ ' ⁇ mice on day 19.
  • FIG. 12 Decreased IFN- ⁇ production in skin lesions of IL-2r A mice. Mice received a topical application of 1.6 % of oxazolone (W/V) on day 1 and challenged with 0.8% of oxazolone on day 8. IFN- ⁇ levels in homogenized ear tissues were measured 24 hrs post- challenge. Each data point represents the mean ⁇ SEM of eight mice. Differences between WT and IL-21 ⁇ ' ⁇ mice were tested using student's t test (*/? ⁇ 0.05). ,
  • FIG. 13 Reduction in T cell proliferation in IL-21-/- mice. Draining lymph nodes were removed immediately after the assessment of ear swelling response 48 hr post oxazolone challenge. T cell proliferation was determined by incubating draining LN cells in the presence of anti-CD3 and anti-CD28 for 72 hrs. Cell proliferation was measured as counts per second using ATP lite assay. Empty bar: no stimulation, solid bar: anti-CD3+anti-CD28 in vitro treatment. Each data point represents the mean + SEM of eight mice. Differences between WT and IL-2r A mice were tested using student's t test (*p ⁇ 0.05).
  • FIG. 14 Reduced Th2 and pro-inflammatory cytokine production pattern in IL-21 '7" mice. Animals were sensitized and challenged with oxazolone. Draining LN cells from the mice were prepared and supernatants were collected. Cytokine production in the supernatant was detected by LINCOplexTM mouse cytokine/chemokine immunoassay kit. Each data point represents the mean ⁇ SEM of eight mice. Differences between WT and IL-21 ⁇ ' ⁇ mice were tested using student's t test (* ⁇ 0.05).
  • FIG. 15 Anti-mIL-21 mAb inhibits the CHS response to oxazolone.
  • BALB/c mice were injected i.p. with different doses of anti-IL-21 mAb prior to receiving a topical application of 1.6 % of oxazolone on day 1.
  • FIG. Enhanced ear swelling in IL-21 "A mice over time. Mice were sensitized with 20 ⁇ L of a 1% (w/v) solution of FITC in 50:50 (vol/vol) acetone dibutylphtalate (DBT) on both sides of the ears on day 1, the mice were challenged on both sides of the ears with 20 ⁇ l of a 0.5% (w/v) solution of FITC in 50:50 acetone DBT on day 8. Each data point represents the mean ⁇ SEM of ten mice. Differences between WT and IL-21 ' ' " mice were tested using student's t test (*/ ⁇ 0.05).
  • DBT acetone dibutylphtalate
  • FIG. Histopathological changes of ear lesions induced by FITC.
  • WT and IL-21 " ' " mice were challenged with control acetone DBT or FITC according to protocol above. Skin specimens were taken at 24 hr and 48 hr after control acetone DBT or FITC challenge and fixed in 10% neutral formalin. Sections were stained with hematoxylin and eosin.
  • A WT 24 hr post control acetone DBT challenge
  • B IL-21 " ' " mice 24 hr post control acetone DBT challenge
  • C C
  • FIG. 19 Elevated serum IgE level in FITC-challenged IL-21 " ⁇ " mice.Animals were sensitized and challenged with FITC. Serum IgE level was measured 72 hr post FITC challenge. Each data point represents the mean ⁇ SEM of ten mice. Differences between WT and IL-21"/" mice were tested using student's t test (*/? ⁇ 0.05).
  • FIG. 20 Enhanced eotaxin production in skin lesions of FITC challenged IL-21 " /" mice. Animals were sensitized and challenged with FITC. Eotaxin level in homogenized ear tissues was measured at indicated time points. Each data point represents the mean ⁇ SEM of ten mice. Differences between WT and IL-21"/" mice were tested using student's t test (*p ⁇ 0.05).
  • FIG. 21 Increased cytokine and chemokine production in skin lesions of FITC challenged IL-21"/ " mice. Animals were sensitized and challenged with FITC. Cytokines and cheniokines in homogenized ear tissues were measured at the indicated time points. Each data point represents the mean + SEM of ten mice. Differences between WT and IL-21 ⁇ ' ⁇ mice were tested using student's t test (*/? ⁇ 0.05).
  • FIG. 22 Enhanced T cell proliferation in FTTC challenged IL-21"/" mice. Draining lymph nodes were removed immediately after the assessment of ear swelling response 24 hr post FITC challenge. T cell proliferation was determined by incubating draining LN cells in the presence of anti-CD3 for 72 hrs. Cell proliferation was measured as counts per second using ATP lite. Empty bar: no stimulation, solid bar: anti-CD3 in vitro treatment. Each data point represents the mean ⁇ SEM of ten mice. Differences between WT and IL-21 " ' ' mice were tested using student's t test (*p ⁇ 0.05). [61] Figure 23. Enhanced Th2 and pro-inflammatory cytokine production in FITC challenged IL-21"/" mice.
  • the present invention provides IL-21 agonists for the treatment of IL-21 related pathologies.
  • the present invention further provides compositions, formulations, methods, devices and uses of such IL-21 agonists, including for therapeutic and diagnostic uses.
  • the present invention further provides agonists that are suitable for treating at least one IL-21 related condition by agonizing IL-21 binding to one or more of its receptors.
  • the present invention provides isolated, recombinant and/or synthetic IL-21 agonists, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding at least one IL-21 agonist.
  • Such agonists of the present invention comprise specific full length agonist sequences, domains, fragments and specified variants thereof, and methods of making and using said nucleic acids and agonists, including therapeutic compositions, methods and devices.
  • IL-21 agonist As used herein, a "IL-21 agonist,” “IL-21 agonist portion,” “IL-21 agonist fragment,” “IL-21 agonist variant” “IL-21 agonist,” “IL-21 agonist portion,” or “IL-21 agonist fragment” and/or “IL-21 agonist variant” and the like activates, enhances, induces, agonizes, or increases at least one IL-21 activity, binding or IL-21 receptor activity or binding in vitro, in situ and/or preferably in vivo, as known in the art.
  • a suitable IL-21 agonist of the present invention can bind at least one IL- 21 protein or receptor and includes IL-21 agonists, antigen-binding fragments thereof, and specified portions, variants or domains thereof that bind specifically to IL-21.
  • a suitable IL-21 agonist, specified portion, or variant can also increase, enhance, induce, activate or agonize IL- 21 protein RNA, DNA or protein synthesis, IL-21 protein release, IL-21 protein or receptor signaling, membrane IL-21 protein cleavage, IL-21 related activity, IL-21 protein production and/or synthesis, e.g., as described herein or as known in the art.
  • IL-21 agonists also termed IL-21 agonists
  • useful in the methods and compositions of the present invention are characterized by high affinity binding to human IL-21, and optionally and preferably having low toxicity.
  • an agonist, specified fragment or variant of the invention, where the individual components, such as the variable region, constant region and framework, individually and/or collectively, optionally and preferably possess low immunogenicity is useful in the present invention.
  • the agonists that can be used in the invention are optionally characterized by their ability to treat patients for extended periods with good to excellent alleviation of symptoms and low toxicity. Low immunogenicity and/or high affinity, as well as other suitable properties, may contribute to the therapeutic results achieved.
  • Low immunogenicity is defined herein as raising significant HAHA, HACA or HAMA responses in less than about 75%, or preferably less than about 50% of the patients treated and/or raising low titres in the patient treated (less than about 300, preferably less than about 100 measured with a double antigen enzyme immunoassay) (Elliott et al., Lancet 344: 1125- 1127 (1994), each of the above references entirely incorporated herein by reference.
  • the isolated nucleic acids of the present invention can be used for production of at least one IL- 21 agonist, which can be used to effect in an cell, tissue, organ or animal (including mammals and humans), to modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of, at least one IL-21 condition, e.g., those associated with IgE production or over production, or
  • a method can comprise administering an effective amount of a composition or a pharmaceutical composition comprising at least one IL-21 agonist to a cell, tissue, organ, animal or patient in need of such modulation, treatment, alleviation, prevention, or reduction in symptoms, effects or mechanisms.
  • the effective amount can comprise an amount of about
  • agonist is intended to encompass agonists, digestion fragments, specified portions and variants thereof, including agonist mimetics or comprising portions of agonists that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain agonists and fragments thereof, and is also is intended to encompass proteins that contain mimetics to therapeutic proteins, antibodies, and digestion fragments, specified portions and variants thereof, wherein the protein comprises at least one functional IL- 21 protein ligand binding region (LBR) that optionally replaces at least one complementarity determining region (CDR) of the antibody from which the IL-21 agonist is derived.
  • LBR functional IL- 21 protein ligand binding region
  • CDR complementarity determining region
  • the agonist comprises at least one CDR or target binding region that specifically binds at least one biologically active IL-21 agonist target, e.g., IL-21 receptor, and further comprises at least 10 to 384-500 amino acids of at least one of SEQ ID NOS: 1-41, or at least a portion of at least one region of a corresponding heavy or light chain amino acid sequence as described in Table 1, optionally further comprising at least one substitution, insertion or deletion as described in Figures 1-41 of publication WO 05/33029 and US serial No. 10/872,932, filed 06/21/2004, entirely incorporated herein by reference.
  • Such IL-21 IgG derived proteins, specified portions or variants include those that mimic the structure and/or function of at least one IL-21 protein antagonist, such as a IL-21 protein antibody or receptor or ligand protein, or fragment or analog.
  • Functional fragments include antigen-binding fragments that bind to human
  • IL-21 receptor proteins or fragments thereof agonist fragments capable of binding to human IL-21 receptor proteins or fragments thereof, including, but not limited to Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab') 2 (e.g., by pepsin digestion), fabc (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the invention (see, e.g., Colligan, Immunology, supra).
  • Fab e.g., by papain digestion
  • Fab' e.g., by pepsin digestion and partial reduction
  • F(ab') 2 e.g., by pepsin digestion
  • Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein, agonists can also be produced in a variety of truncated forms using agonist genes in which one or more stop codons have been introduced upstream of the natural stop site.
  • a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CHi domain and/or hinge region of the heavy chain.
  • the various portions of agonists can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
  • a nucleic acid encoding the variable and constant regions of a human agonist chain can be expressed to produce a contiguous protein. See, e.g., Colligan, Immunology, supra, sections 2.8 and 2.10, for fragmentation and Ladner et al, U.S. Patent No. 4,946,778 and Bird, R.E. et al, Science, 242: 423-426 (1988), regarding single chain agonists, each of which publications are entirely incorporated herein by reference.
  • IL-21 antibody or antibody fusion agonist refers to an agonist in which substantially every part of the protein (e.g., in agonist antibodies,, CDR, LBR, framework, C L , C ⁇ domains (e.g., C H 1, C H 2, C H 3), hinge, (V L , V H )) is substantially non- immunogenic, with only minor sequence changes or variations. Such changes or variations optionally and preferably retain or reduce the immunogenicity in humans relative to non- modified human agonists.
  • a human agonist is distinct from a chimeric or humanized Ig.
  • a human agonist can be produced by a non-human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes.
  • a human agonist when a human agonist is a single chain agonist, it can comprise a linker peptide that is not found in native human agonists.
  • an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
  • linker peptides are considered to be of human origin.
  • IL-21 agonists that comprise at least one EL-21 protein ligand or receptor thereof can be designed against an appropriate ligand, such as isolated and/or IL-21 protein, or a portion thereof (including synthetic molecules, such as synthetic peptides). Preparation of such IL-21 agonists are performed using known techniques to identify and characterize ligand binding regions or sequences of at least one IL-21 protein or portion thereof.
  • MU-I is homologous to the shared beta chain of the DL-2 and IL-15 receptors, and IL-4 alpha. (Ozaki et al. (2000) supra).
  • IL-21R/MU-1 Upon ligand binding, IL-21R/MU-1 is capable of interacting with a common gamma cytokine receptor chain (gamma c) (Asao et al. (2001) J. Immunol. 167:1-5), and inducing the phosphorylation of STATl and STAT3 (Asao et al. (2001) or STAT5 (Ozaki et al. (2000).
  • MU- 1 shows widespread lymphoid tissue distribution.
  • the term "MU-I" refers to a receptor (preferably of mammalian, e.g., murine or human origin) which is capable of interacting with, e.g., binding to, JL-21 (preferably of mammalian, e.g., murine or human IL-21) and having one of the following features: (i) an amino acid sequence of a naturally occurring mammalian MU-I polypeptide IL-21R/MU-1 or a fragment thereof, e.g., an amino acid sequence shown as SEQ ID NO:43 (human) or a fragment thereof; (ii) an amino acid sequence substantially homologous to, e.g., at least 85%, 90%, 95%, 98%, 99% homologous to, an amino acid sequence shown as SEQ ID NO:43 or a fragment thereof; ( ⁇ i) an amino acid sequence which is encoded by a naturally occurring mammalian IL-21 R/MU-1 nucleotide sequence or
  • the IL-21R/MU-1 is of mammalian, preferably, human origin. Analysis of the human IL- 21R/MU-1 amino acid sequence revealed the following structural features: a leader sequence from about (about amino acids 1-19 of SEQ ID NO:43; WSXWS motif (about amino acids 213- 217 of SEQ ID NO:43); transmembrane domain (about amino acids 236-252 of SEQ ID NO:43); an extracellular domain from about amino acids 1-235 of SEQ ID NO:43; and an intracellular domain from about 253-538 of SEQ ID NO:43.
  • the mature human IL-21R/MU-1 is believed to have the sequence of amino acids 20-538 of SEQ ID NO:43.
  • the IL-21R/MU-1 cDNA was deposited with the American Type Culture Collection on Mar. 10, 1998, as accession number ATCC 98687.
  • Human antibody or antibody fusion agonists that are specific for the IL-21 receptor can be raised against an appropriate immunogenic antigen, such as isolated EL-21 protein or a portion thereof (including synthetic molecules, such as synthetic peptides).
  • an appropriate immunogenic antigen such as isolated EL-21 protein or a portion thereof (including synthetic molecules, such as synthetic peptides).
  • Preparation of immunogenic antigens, and monoclonal agonist production can be performed using any suitable technique. A variety of methods have been described (see e.g., Kohler et ah, Nature, 256: 495- 497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et ⁇ l, Nature 266: 550-552 (1977); Koprowski et al, U.S. Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, agonists: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current
  • a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, S ⁇ 2/0-AG14, NSO, NSl, NS2, AE-I, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SSl, Sp2 SA5, U937, MLA 144, ACT W, MOLT4, DA-I , JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or the like, or heteromylomas, fusion products thereof, or any cell or fusion cell derived therefrom, or any other suitable cell line as known in the art, see, e.g., www.
  • a suitable immortal cell line e.g., a myeloma cell line such as, but not limited to, Sp2/0
  • agonist producing cells such as, but not limited to, isolated or cloned spleen cells, or any other cells expressing heavy or light chain constant or variable or framework or CDR sequences, either as endogenous or heterologous nucleic acid, as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and the like or any combination thereof. See, e.g.,
  • Agonist antibody producing cells can be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals that have been immunized with the antigen of interest. Any other suitable host cell can also be used for expressing heterologous or endogenous nucleic acid encoding an agonist, specified fragment or variant thereof, of the present invention.
  • the fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods. Cells which produce agonists with the desired specificity can be selected by a suitable assay (e.g., ELISA).
  • Suitable methods of producing or isolating antibodies or other protein IL-21 agonists of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from Cambridge antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, DE;
  • a peptide or protein library e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from Cambridge antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, DE;
  • SLAM selected lymphocyte antibody method
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain.
  • Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988), each of which is entirely incorporated herein by reference), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • "humanized" agonists are chimeric agonists (Cabilly et al., supra), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized agonists are typically human agonists in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent agonists.
  • the choice of human variable domains, both light and heavy, to be used in making the humanized agonists can be used to reduce antigenicity.
  • the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. MoI. Biol.
  • Another method uses a particular framework derived from the consensus sequence of all human agonists of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized agonists (Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), each of which is entirely incorporated herein by reference).
  • Agonists can also optionally be humanized with retention of high affinity for the antigen and other favorable biological properties.
  • humanized agonists are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.
  • Human monoclonal agonists can be made by the hybridoma method.
  • Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal agonists have been described, for example, by Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol. 147:86 (1991), each of which is entirely incorporated herein by reference.
  • phage display technology as presented above can be used to produce human agonists and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
  • V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell.
  • Phage display can be performed in a variety of formats; for their review see, e.g., Johnson et al., Current Opinion in Structural Biology 3:564 (1993), each of which is entirely incorporated herein by reference.
  • V-gene segments can be used for phage display. Clackson et al., Nature 352:624 (1991) isolated a diverse array of anti-oxazolone agonists from a small random combinatorial library of V genes derived from the spleens of, immunized mice.
  • a repertoire of V genes from unimmunized human donors can be constructed and agonists to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. MoI. Biol. 222:581 (1991), or Griffith et al., EMBO J. 12:725 (1993), each of which is entirely incorporated herein by reference.
  • Bispecific agonists can also be used that are monoclonal, preferably human or humanized, agonists that have binding specificities for at least two different antigens.
  • one of the binding specificities is for at least one IL-21 protein, the other one is for any other antigen.
  • bispecific agonists specifically binding a IL-21 protein and at least one neurotrophic factor, or two different types of IL-21 polypeptides are within the scope of the present invention.
  • Methods for making bispecific agonists are known in the art.
  • bispecific agonists are 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 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829 published 13 May 1993, and in Traunecker et al., EMBO J.
  • antibody- variable domains with the desired binding specificities are fused to immunoglobulin constant-domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, the second heavy chain constant region (C sub H2), and the third heavy chain constant region (C sub H 3). It is preferred to have the first heavy-chain constant region (C sub Hl), 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. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the production of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
  • the bispecific agonists are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm.
  • This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation.
  • Heteroconjugate agonists are also within the scope of the present invention.
  • Heteroconjugate agonists are composed of two covalently joined agonists. Such agonists 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 92/00373; and EP 03089).
  • Heteroconjugate agonists can be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • At least one IL-21 agonist of the present invention is produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells.
  • Immortalized IL-21 producing cells can be produced using suitable methods, for example, fusion of a human agonist-producing cell and a heteromyeloma, or immortalization of an activated human B cell via infection with Epstein Barr virus (Niedbala et al, Hybridoma, i7(3):299-304 (1998); Zanella et al, J Immunol Methods, 25 ⁇ 5(2):205-215 (1992); Gustafsson et al, Hum agonists Hybridomas, 2(1)26-32 (1991)) .
  • the human anti-human IL-21 proteins or fragments is generated by immunization of a transgenic animal (e.g., mouse, rat, hamster, non-human primate, and the like) capable of producing a repertoire of human agonists, as described herein and/or as known in the art.
  • a transgenic animal e.g., mouse, rat, hamster, non-human primate, and the like
  • Cells that produce a human IL- 21 agonist can be isolated from such animals and immortalized using suitable methods, such as the methods described herein.
  • Transgenic mice that can produce a repertoire of human agonists that bind to human antigens can be produced by known methods (e.g., but not limited to, U.S. Pat. Nos: 5,770,428, 5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al; Jakobovits et al WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg et al. WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al.
  • known methods e.g., but not limited to, U.S. Pat. Nos: 5,770,428, 5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al
  • mice comprise at least one transgene comprising DNA from at least one human immunoglobulin locus that is functionally rearranged, or which can undergo functional rearrangement.
  • the endogenous immunoglobulin loci in such mice can be disrupted or deleted to eliminate the capacity of the animal to produce agonists encoded by endogenous genes.
  • the term "functionally rearranged,” as used herein refers to a segment of DNA from an immunoglobulin locus that has undergone V(D)J recombination, thereby producing an immunoglobulin gene that encodes an immunoglobulin chain (e.g., heavy chain, light chain), or any portion thereof.
  • a functionally rearranged immunoglobulin gene can be directly or indirectly identified using suitable methods, such as, for example, nucleotide sequencing, hybridization (e.g., Southern blotting, Northern blotting) using probes that can anneal to coding joints between gene segments or enzymatic amplification of immunoglobulin genes (e.g., polymerase chain reaction) with primers that can anneal to coding joints between gene segments.
  • Whether a cell produces an agonist comprising a particular variable region or a variable region comprising a particular sequence can also be determined using suitable methods.
  • mRNA can be isolated from an agonist- producing cell (e.g., a hybridoma or recombinant cell or other suitable source) and used to produce cDNA encoding the agonist thereof.
  • the cDNA can be cloned and sequenced or can be amplified (e.g., by polymerase chain reaction or other known and suitable methods) using a first primer that anneals specifically to a portion of the variable region of interest (e.g., CDR, coding joint) and a second primer that anneals specifically to non- variable region sequences (e.g., C H 1,
  • Screening agonists for specific binding to similar proteins or fragments can be conveniently achieved using peptide display libraries. This method involves the screening of large collections of peptides for individual members having the desired function or structure, agonist screening of peptide display libraries is well known in the art.
  • the displayed peptide sequences can be from 3 to
  • Agonists, specified portions and variants of the present invention can also be prepared using at least one IL-21 agonist encoding nucleic acid to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that produce such agonists in their milk.
  • transgenic animals or mammals such as goats, cows, horses, sheep, and the like, that produce such agonists in their milk.
  • Such animals can be provided using known methods. See, e.g., but not limited to, US patent nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489, and the like, each of which is entirely incorporated herein by reference.
  • Agonists of the present invention can additionally be prepared using at least one IL-21 agonist encoding nucleic acid to provide transgenic plants and cultured plant cells (e.g., but not limited to tobacco and maize) that produce such agonists, specified portions or variants in the plant parts or in cells cultured therefrom.
  • transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large amounts of recombinant proteins, e.g., using an inducible promoter. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) and references cited therein.
  • transgenic maize have been used to express mammalian proteins at commercial production levels, with biological activities equivalent to those produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al., Adv. Exp. Med. Biol. 464:127-147 (1999) and references cited therein.
  • Agonists have also been produced in large amounts from transgenic plant seeds including agonist fragments, such as single chain agonists (scFv's), including tobacco seeds and potato tubers. See, e.g., Conrad et al., Plant MoI. Biol. 38:101-109 (1998) and reference cited therein.
  • agonists, specified portions and variants of the present invention can also be produced using transgenic plants, according to know methods. See also, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (Oct., 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans. 22:940-944 (1994); and references cited therein. Each of the above references is entirely incorporated herein by reference.
  • the agonists of the invention can bind human IL-21 proteins or fragments with a wide range of affinities (K D ).
  • at least one human antibody or fragment of the present invention can optionally bind human IL-21 proteins or fragments with high affinity.
  • a human antibody or fragment can bind human IL-21 proteins or fragments with a K D equal to or less than about 10 '9 M or, more preferably, with a K D equal to or less than about 0.1-9.9 (or any range or value therein) X 10 "10 M, 10 "n , 10 "12 , 10 "13 or any range or value therein.
  • the affinity or avidity of an agonist for an antigen can be determined experimentally using any suitable method.
  • any suitable method See, for example, Berzofsky, et al, "agonist- Antigen Interactions," In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, NY (1992); and methods described herein).
  • the measured affinity of a particular agonist-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH).
  • affinity and other antigen-binding parameters e.g., K D , K a , Ka
  • K D , K a , Ka are preferably made with standardized solutions of agonist and antigen, and a standardized buffer, such as the buffer described herein.
  • nucleic acid molecule of the present invention encoding at least one IL-21 agonist can be obtained using methods described herein or as known in the art.
  • Nucleic acid molecules of the present invention can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combinations thereof.
  • the DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.
  • Isolated nucleic acid molecules of the present invention can include nucleic acid molecules comprising an open reading frame (ORF), optionally with one or more introns, e.g., but not limited to, at least one specified portion of at least one CDR, as CDRl, CDR2 and/or CDR3 of at least one heavy chain or light chain, respectively; nucleic acid molecules comprising the coding sequence for a IL-21 agonist; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode at least one IL-21 agonist as described herein and/or as known in the art.
  • ORF open reading frame
  • introns e.g., but not limited to, at least one specified portion of at least one CDR, as CDRl, CDR2 and/or CDR3 of at least one heavy chain or light chain, respectively
  • nucleic acid molecules comprising the coding sequence for a IL-21 agonist
  • nucleic acid variants that code for specific IL-21 agonists of the present invention. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are included in the present invention.
  • nucleic acid molecules of the present invention which comprise a nucleic acid encoding a IL-21 agonist can include, but are not limited to, those encoding the amino acid sequence of an agonist fragment, by itself; the coding sequence for the entire agonist or a portion thereof; the coding sequence for an agonist, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional, non-coding sequences, including but not limited to, non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example - ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities.
  • the sequence encoding an agonist can be fused to
  • the present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide encoding an IL-21 agonist of the present invention.
  • the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides.
  • polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library.
  • the polynucleotides are genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic acid library.
  • the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences.
  • the cDNA libraries can be normalized to increase the representation of rare sequences.
  • Low or moderate stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences.
  • Moderate and high stringency conditions can optionally be employed for sequences of greater identity.
  • Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences.
  • polynucleotides of this invention will encode at least a portion of an agonist encoded by the polynucleotides described herein.
  • the polynucleotides of this invention embrace nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding an agonist of the present invention. See, e.g., Ausubel, supra; Colligan, supra, each entirely incorporated herein by reference.
  • the isolated nucleic acids of the present invention can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, as well- known in the art.
  • the nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present invention.
  • a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide.
  • translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the present invention.
  • a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention.
  • the nucleic acid of the present invention - excluding the coding sequence - is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention.
  • Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell.
  • Use of cloning vectors, expression vectors, adapters, and Linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra) [110] Recombinant Methods for Constructing Nucleic Acids
  • RNA, cDNA, genomic DNA, or any combination thereof can be obtained from biological sources using any number of ! cloning methodologies known to those of skill in the art.
  • oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library.
  • the isolation of RNA, and construction of cDNA and genomic libraries, is well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra)
  • a cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention, such as those disclosed herein. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms.
  • Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms.
  • degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur.
  • the degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide.
  • the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50%.
  • the degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium.
  • the degree of complementarity will optimally be 100%, or 90-100%, or any range or value therein.
  • minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium.
  • RNA or DNA Methods of amplification of RNA or DNA are well known in the art and can be used according to the present invention without undue experimentation, based on the teaching and guidance presented herein.
  • Known methods of DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis, et al.; 4,795,699 and
  • PCR polymerase chain reaction
  • in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
  • examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et al., U.S. Patent No. 4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, CA (1990).
  • kits for genomic PCR amplification are known in the art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).
  • the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.
  • the isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.
  • Chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences.
  • the present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention.
  • a nucleic acid sequence of the present invention for example a cDNA or a genomic sequence encoding an agonist of the present invention, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell.
  • a recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention.
  • isolated nucleic acids that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in intron) of a non-heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention.
  • endogenous promoters can be altered in vivo or in vitro by mutation, deletion and/or substitution.
  • a polynucleotide of the present invention can be expressed in either sense or anti-sense orientation as desired. It will be appreciated that control of gene expression in either sense or anti- sense orientation can have a direct impact on the observable characteristics. [122] Another method of suppression is sense suppression. Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes.
  • a variety of cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect and/or cleave nucleic acids.
  • the present invention also relates to vectors that include isolated nucleic acid molecules of the present invention, host cells that are genetically engineered with the recombinant vectors, and the production of at least one IL-21 agonist by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.
  • the polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. [127] The DNA insert should be operatively linked to an appropriate promoter.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.
  • a termination codon e.g., UAA, UGA or UAG
  • Expression vectors will preferably but optionally include at least one selectable marker.
  • markers include, e.g., but not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, US Pat. Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017, ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase (GS, US Pat. Nos.
  • At least one agonist of the present invention can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of an agonist to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to an agonist of the present invention to facilitate purification. Such regions can be removed prior to final preparation of an agonist or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.
  • nucleic acids of the present invention can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding an agonist of the present invention.
  • Such methods are well known in the art, e.g., as described in US patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.
  • Illustrative of cell cultures useful for the production of the agonists, specified portions or variants thereof, are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions or bioreactors can also be used.
  • COS-I e.g., ATCC CRL 1650
  • COS-7 e.g., ATCC CRL-1651
  • HEK293, BHK21 e.g., ATCC CRL-10
  • CHO e.g., ATCC CRL 1610
  • BSC-I e.g., ATCC CRL-26 ceU lines
  • Cos-7 cells CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Agl4, 293 cells, HeLa cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas, Va.
  • Preferred host cells include cells of lymphoid origin such as myeloma and lymphoma cells.
  • Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Agl4 cells (ATCC Accession Number CRL-1851).
  • the recombinant cell is a P3X63Ab8.653 or an SP2/0-Agl4 cell.
  • Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (US Pat. Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-I alpha promoter (US Pat. No.
  • polyadenlyation or transcription terminator sequences are typically incorporated into the vector.
  • An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included.
  • An example of a splicing sequence is the VPl intron from SV40 (Sprague, et al., J. Virol.45:773-781 (1983)).
  • gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art.
  • a IL-21 agonist can be recovered and purified from recombinant cell cultures by well- known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be employed for purification.
  • HPLC high performance liquid chromatography
  • Agonists of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells.
  • a eukaryotic host including, for example, yeast, higher plant, insect and mammalian cells.
  • the agonist of the present invention can be glycosylated or can be non-glycosylated, with glycosylated preferred.
  • Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein
  • the isolated agonists of the present invention can comprise an agonist encoded by any one of the polynucleotides of the present invention as discussed more fully herein, or any isolated or prepared agonist thereof.
  • the polypeptide antibody or antibody fusion agonist or antigen-binding fragment binds human IL-21 proteins or fragments and, thereby substantially neutralizes the biological activity of the protein.
  • An agonist, thereof, that partially or preferably substantially neutralizes at least one biological activity of at least one IL-21 protein or fragment can bind the protein or fragment and thereby inhibit activities mediated through the binding of IL-21 to at least one IL-21 receptor or through other IL-21-dependent or mediated mechanisms.
  • neutralizing agonist refers to an agonist that can inhibit human p40 or pl9 protein or fragment related-dependent activity by about 20-120%, preferably by at least about 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or more depending on the assay.
  • the capacity of anti- human IL-21 agonist to inhibit human IL-21 related-dependent activity is preferably assessed by at least one suitable IL-21 agonist or protein assay, as described herein and/or as known in the art.
  • a human agonist of the invention can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambda light chain.
  • the human agonist comprises an IgG heavy chain or defined fragment, for example, at least one of isotypes, IgGl, IgG2, IgG3 or IgG4.
  • Agonists of this type can be prepared by employing a transgenic mouse or other transgenic non-human mammal comprising at least one human light chain (e.g., IgG, IgA and IgM (e.g., ⁇ l, ⁇ 2, ⁇ 3, ⁇ 4) transgenes as described herein and/or as known in the art.
  • the anti-human IL-21 agonist thereof comprises an IgGl heavy chain and an IgGl light chain.
  • At least one agonist of the invention binds at least one specified epitope specific to at least one IL-21 receptor or ligand, portion or any combination thereof.
  • the at least one epitope can comprise at least one agonist binding region that comprises at least one portion of said protein, which epitope is preferably comprised of at least one extracellular, soluble, hydrophillic, external or cytoplasmic portion of said protein.
  • a IL-21 agonist specifically binds at least one epitope comprising at least 1-3, to the entire amino acid sequence, selected from the group consisting of at least one subunit of human IL-21 receptor (e.g., SEQ ID NO.43).
  • the at least one specified epitope can comprise any combination of at least one amino acid of the IL-21 receptor or ligand, such as but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acids of at least one of, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140- 150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240- 250, 250-260, 260-270, 280-290, 290-300, 300-306, of SEQ ID NO:43.
  • the human agonist or antigen-binding fragment of the present invention will comprise an antigen-binding region that comprises at least one human complementarity determining region (CDRl, CDR2 and CDR3) or variant of at least one heavy chain variable region and at least one human complementarity determining region (CDRl, CDR2 and CDR3) or variant of at least one light chain variable region.
  • the agonist or antigen-binding portion or variant can comprise at least one of the heavy chain CDR3, and/or a light chain CDR3.
  • the agonist or antigen-binding fragment can have an antigen-binding region that comprises at least a portion of at least one heavy chain CDR (i.e., CDRl, CDR2 and/or CDR3) having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3.
  • the agonist or antigen-binding portion or variant can have an antigen-binding region that comprises at least a portion of at least one light chain CDR (i.e., CDRl, CDR2 and/or CDR3) having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3.
  • Such agonists can be prepared by chemically joining together the various portions (e.g., CDRs, framework) of the agonist using conventional techniques, by preparing and expressing a (i.e., one or more) nucleic acid molecule that encodes the agonist using conventional techniques of recombinant DNA technology or by using any other suitable method.
  • the IL-21 agonist can comprise at least one of a heavy or light chain variable region having a defined amino acid sequence.
  • the IL-21 agonist comprises at least one of at least one heavy chain variable region and/or at least one light chain variable region.
  • Human agonists that bind to human IL-21 proteins or fragments and that comprise a defined heavy or light chain variable region can be prepared using suitable methods, such as phage display (Katsube, Y., et al, IntJMol. Med, l(5):863-868 (1998)) or methods that employ transgenic animals, as known in the art and/or as described herein.
  • a transgenic mouse comprising a functionally rearranged human immunoglobulin heavy chain transgene and a transgene comprising DNA from a human immunoglobulin light chain locus that can undergo functional rearrangement, can be immunized with human IL-21 proteins or fragments thereof to elicit the production of agonists.
  • the agonist producing cells can be isolated and hybridomas or other immortalized agonist-producing cells can be prepared as described herein and/or as known in the art.
  • the agonist, specified portion or variant can be expressed using the encoding nucleic acid or portion thereof in a suitable host cell.
  • the invention also relates to agonists, antigen-binding fragments, immunoglobulin chains and CDRs comprising amino acids in a sequence that is substantially the same as an amino acid sequence described herein.
  • agonists or antigen-binding fragments and agonists comprising such chains or CDRs can bind human IL-21 proteins or fragments with high affinity (e.g., K 0 less than or equal to about 10 '9 M).
  • Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.
  • a conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/ hydrophilicity) that are similar to those of the first amino acid.
  • Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (T), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.
  • Amino Acid Codes K, arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E;
  • amino acids that make up IL-21 agonists of the present invention are often abbreviated.
  • the amino acid designations can be indicated by designating the amino acid by its single letter code, its three letter code, name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994):
  • An EL-21 agonist of the present invention can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein.
  • the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions, insertions or deletions for any given IL-21 polypeptide will not be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, as specified herein.
  • Amino acids in a IL-21 agonist of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine- scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to at least one IL-21 neutralizing activity. Sites that are critical for agonist binding can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. MoI. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).
  • the agonists of the present invention can comprise any number of contiguous amino acid residues from an agonist of the present invention, wherein that number is selected from the group of integers consisting of from 10-100% of the number of contiguous residues in an IL-21 agonist.
  • this subsequence of contiguous amino acids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids in length, or any range or value therein.
  • the present invention includes at least one biologically active agonist of the present invention.
  • Biologically active agonists have a specific activity at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-1000% of that of the native (non-synthetic), endogenous or related and known agonist. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those of skill in the art.
  • the invention relates to human agonists and antigen-binding fragments, as described herein, which are modified by the covalent attachment of an organic moiety.
  • modification can produce an agonist or antigen-binding fragment with improved pharmacokinetic properties (e.g., increased in vivo serum half-life).
  • the organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group.
  • the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.
  • a polyalkane glycol e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)
  • carbohydrate polymer e.g., amino acid polymer or polyvinyl pyrolidone
  • the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.
  • the modified agonists and antigen-binding fragments of the invention can comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the agonist.
  • Each organic moiety that is bonded to an agonist or antigen-binding fragment of the invention can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group.
  • fatty acid encompasses mono-carboxylic acids and di- carboxylic acids.
  • Hydrophilic polymers suitable for modifying agonists of the invention can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone.
  • polyalkane glycols e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like
  • carbohydrates e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like
  • polymers of hydrophilic amino acids e.g., polylys
  • the hydrophilic polymer that modifies the agonist of the invention has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity.
  • PEG 5O oo and PEG 2 o,ooo wherein the subscript is the average molecular weight of the polymer in Daltons, can be used.
  • the hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods.
  • a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N, N-carbonyl d ⁇ midazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.
  • an activated carboxylate e.g., activated with N, N-carbonyl d ⁇ midazole
  • Fatty acids and fatty acid esters suitable for modifying agonists of the invention can be saturated or can contain one or more units of unsaturation.
  • Fatty acids that are suitable for modifying agonists of the invention include, for example, n-dodecanoate (C ]2 , laurate), n- tetradecanoate (C H , myristate), n-octadecanoate (Ci 8 , stearate), n-eicosanoate (C 2 o, arachidate), n-docosanoate (C 22 , behenate), n-triacontanoate (C 30 ), n-tetracontanoate (C 40 ), czs- ⁇ 9- octadecanoate (C 18 , oleate), all c ⁇ - ⁇ 5, 8, 11, 14-eicosatetraenoate (C 2 o, arachidonate), octanedioic acid,
  • the modified human agonists and antigen-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents.
  • An "activating group” is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agent and the second chemical group.
  • amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, tluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and 5 the like.
  • Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like.
  • An aldehyde functional group can be coupled to amine- or hydrazide-containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages.
  • Suitable methods to introduce activating groups 0 into molecules are known in the art (see for example, Hermanson, G. T., Bioconjugate
  • An activating group can be bonded directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester), or through a linker moiety, for example a divalent C 1 -Cn group wherein one or more carbon atoms can be replaced by a heteroatom such as oxygen, nitrogen or sulfur.
  • Suitable linker moieties include, 5 for example, tetraethylene glycol, -(CHa) 3 -, -NH-(CH 2 ) 6 -NH-, -(CH 2 J 2 -NH- and -CH 2 -O-CH 2 -
  • Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono- Boc-diaminohexane) with a fatty acid in the presence of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid O carboxylate.
  • a mono-Boc-alkyldiamine e.g., mono-Boc-ethylenediamine, mono- Boc-diaminohexane
  • EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • the Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described, or can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimido derivative of the fatty acid.
  • TFA trifluoroacetic acid
  • the modified agonists of the invention can be produced by reacting a human agonist or antigen-binding fragment with a modifying agent.
  • the organic moieties can be bonded to the agonist in a non-site specific manner by employing an amine-reactive modifying agent, for example, an NHS ester of PEG.
  • Modified human agonists or antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intra-chain disulfide bonds) of an agonist or antigen-binding fragment. The reduced agonist or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified agonist of the invention.
  • Modified human agonists and antigen-binding fragments comprising an organic moiety that is bonded to specific sites of an agonist of the present invention can be prepared using suitable methods, such as reverse proteolysis (Fisch etah, Bioconjugate Chem., 3:147-153 (1992); Werlen et al, Bioconjugate Chem., 5:411-417 (1994); Kumaran et al, Protein ScL 6(10):2233-2241 (1997); Itoh et al, Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al, Biotechnol. Bioeng., 56(4):456-463 (1997)), and the methods described in Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996).
  • suitable methods such as reverse proteolysis (Fisch etah, Bioconjugate Chem., 3:147-153 (1992); Werlen et al, Bioconjugate Chem., 5:411-4
  • the present invention also provides at least one IL-21 agonist composition comprising at least one, at least two, at least three, at least four, at least five, at least six or more IL-21 agonists thereof, as described herein and/or as known in the art that are provided in a non- naturally occurring composition, mixture or form.
  • Such compositions comprise non-naturally occurring compositions comprising at least one or two full length, C- and/or N-terminally deleted variants, domains, fragments, or specified variants, of the IL-21 agonist amino acid sequence, or specified fragments, domains or variants thereof.
  • Such composition percentages are by weight, volume, concentration, molarity, or molality as liquid or dry solutions, mixtures, suspension, emulsions or colloids, as known in the art or as described herein.
  • IL-21 agonist compositions of the present invention can further comprise at least one of any suitable auxiliary, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like.
  • Pharmaceutically acceptable auxiliaries are preferred.
  • Non-limiting examples of, and methods of preparing such sterile solutions are well known in the art, such as, but limited to, Gennaro, Ed., Remington 's Pharmaceutical Sciences, 18 th Edition, Mack Publishing Co. (Easton, PA) 1990.
  • Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the IL-21 composition as well known in the art or as described herein.
  • compositions include but are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • amino acid/agonist components which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • One preferred amino acid is glycine.
  • Carbohydrate excipients suitable for use in the invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myoinositol and the like.
  • monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
  • disaccharides such as lactose, sucrose, trehalose,
  • IL-21 agonist compositions can also include a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base.
  • Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers.
  • Preferred buffers for use in the present compositions are organic acid salts such as citrate.
  • the IL-21 agonist compositions of the invention can include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl- ⁇ -cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).
  • polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl- ⁇ -cyclodextrin), polyethylene glycols
  • compositions comprising further therapeutic components
  • the composition 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 drag, an antineoplastic, an immunomodulation drag, an ophthalmic, otic or nasal drug, a topical drag, a nutritional drag or the like.
  • CV cardiovascular
  • CNS central nervous system
  • ANS autonomic nervous system
  • GI gastrointestinal
  • a hormonal drug a drug for fluid or electrolyte balance
  • a hematologic drag an antineoplastic
  • an immunomodulation drag an ophthalmic, otic or nasal drug
  • topical drag a nutritional drag or the like.
  • Drags are well known in the art, including formulations, indications, dosing and administration for each presented herein (see., e.g., Nursing 2001 Handbook of Drags, 21 st edition, Springhouse Corp., Springhouse, PA, 2001; Health Professional's Drag Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper 1 Saddle River, NJ; Pharmcotherapy Handbook, Wells et al., ed., Appleton & Lange, Stamford, CT, each entirely incorporated herein by reference).
  • the respiratory tract drag can be at least one selected from antihistamines, bronchodilators, expectorants or at least one anti-tussives, miscellaneous respiratory drags.
  • the GI tract drag can be at least one selected from antacids or at least one adsorbents or at least one antiflatulents, digestive enzymes or at least one gallstone solubilizers, antidiarrheals, laxatives, anti-emetics, antiulcer drags.
  • the hormonal drag can be at least one selected from corticosteroids, androgens or at least one anabolic steroids, estrogens or at least one progestins, gonadotropins, anti-diabetic drags or at least one glucagon, thyroid hormones, thyroid hormone antagonists, pituitary hormones, parathyroid-like drags.
  • the drag for fluid and electrolyte balance can be at least one selected from diuretics, electrolytes or at least one replacement solutions, acidifiers or at least one alkalinizers.
  • the anti-neoplasties can be at least one selected from alkylating drags, antimetabolites, antibiotic antineoplastics, antineoplastics that alter hormone balance, miscellaneous antineoplastics.
  • the immunomodulation drag can be at least one selected from immunosuppressants, vaccines or at least one toxoids, antitoxins or at least one antivenins, immune serums, biological response modifiers.
  • the ophthalmic, otic, and nasal drags can be at least one selected from ophthalmic anti-infectives, ophthalmic antiinflammatories, miotics, mydriatics, ophthalmic vasoconstrictors, miscellaneous ophthalmics, otics, nasal drags.
  • the topical drag can be at least one selected from local anti-infectives, scabicides or at least one pediculicides, topical corticosteroids.
  • the nutritional drag can be at least one selected from vitamins, minerals, or calorics. See, e.g., contents of Nursing 2001 Drug
  • the at least one antituberculotic or antileprotic can be at least one selected from clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, streptomycin sulfate.
  • the at least one aminoglycoside can be at least one selected from amikacin sulfate, gentamicin sulfate, neomycin sulfate, streptomycin sulfate, 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, ticarcillin disodium/clavulanate potassium.
  • the at least one cephalosporin can be at least one selected from at least one of cefaclor, cefadroxil, cefazolin sodium, cefdinir, cefepime hydrochloride, cefixime, cefmetazole 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, 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, tetracycline hydrochloride.
  • the at least one sulfonamide can be at least one selected from co-trimoxazole, sulfadiazine, sulfamethoxazole, sulfisoxazole, 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, trovafloxacin 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, trovafloxacin 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, nelf ⁇ navir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, 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, 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, vancomycin hydrochloride. (See, e.g., pp. 24-214 of Nursing 2001 Drug Handbook.)
  • the at least one nonnarcotic analgesic or antipyretic can be at least one selected from acetaminophen, aspirin, choline magnesium trisalicylate, diflunisal, 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, sulindac.
  • 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, phentermine hydrochloride. (See, e.g., pp. 337-530 of Nursing 2001 Drug Handbook.) [168]
  • the at least one skeletal muscle relaxant can be at least one selected from baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine hydrochloride, dantrolene sodium, methocarbamol, tizanidine hydrochloride.
  • the at least one neuromuscular blockers 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, vecuronium bromide. (See, e.g., pp. 531-84 of Nursing 2001 Drug Handbook.)
  • 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, triprolidine hydrochloride.
  • the at least one bronchodilators 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, theophylline.
  • the at least one expectorants or antitussives can be at least one selected from benzonatate, codeine phosphate, codeine sulfate, dextramethorphan hydrobromide, diphenhydramine hydrochloride, guaifenesin, hydromorphone hydrochloride.
  • the at least one miscellaneous respiratory drug can be at least one selected from acetylcysteine, beclomethasone dipropionate, beractant, budesonide, calfactant, cromolyn sodium, dornase alfa, epoprostenol sodium, flunisolide, fluticasone propionate, montelukast sodium, nedocromil sodium, palivizumab, triamcinolone acetonide, zafirlukast, zileuton. (See, e.g., pp. 585-642 of Nursing 2001 Drug Handbook.)
  • the at least one coricosteroids 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, triamcinolone diacetate.
  • the at least one androgen or anabolic steroids can be at least one selected from danazol, fluoxymesterone, methyltestosterone, nandrolone decanoate, nandrolone phenpropionate, testosterone, testosterone cypionate, testosterone enanthate, testosterone propionate, testosterone transdermal system.
  • the at least one estrogen or progestin can be at least one selected from esterif ⁇ ed 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, e
  • the at least one gonadroptropin can be at least one selected from ganirelix acetate, gonadoreline acetate, histrelin acetate, 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, troglitazone.
  • the at least one pituitary hormone can be at least one selected from corticotropin, cosyntropin, desmopressin acetate, leuprolide acetate, repository corticotropin, somatrem, somatropin, vasopressin.
  • the at least one parathyroid-like drug can be at least one selected from calcifediol, calcitonin (human), calcitonin (salmon), calcitriol, dihydrotachysterol, etidronate disodium. (See, e.g., pp. 696-796 of Nursing 2001 Drug Handbook.)
  • 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, 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), sodium chloride.
  • the at least one acidifier or alkalinizer can be at least one selected from sodium bicarbonate, sodium lactate, tromethamine.
  • 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, 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, 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 HI (human), factor IX (human), factor IX complex, plasma protein fractions.
  • the at least one thrombolytic enzyme can be at least one selected from alteplase, anistreplase, reteplase (recombinant), streptokinase, urokinase. (See, e.g., pp.
  • 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, 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, 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, valrubicin.
  • the at least one antineoplastics that alter 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, 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, vinorelbine tartrate. (See, e.g., pp. 867-963 of Nursing 2001 Drug Handbook)
  • the at least one local anti-infectives 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, naftifme hydrochloride, neomycin sulfate, nitrofurazone, nystatin, silver sulfadiazine, terbinafine hydrochloride, terconazole, tetracycline hydrochloride, tioconazole, tolnaftate.
  • the at least one scabicide or pediculicide can be at least one selected from crotamiton, lindane, permethrin, 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, triamcinolone acetonide. (See, e.g., pp. 1098-1136 of Nursing 2001 Drug Handbook.)
  • 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, zinc.
  • the at least one calorics 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, medium-chain triglycerides. (See, e.g., pp. ' 1137-63 of Nursing 2001 Drug Handbook.)
  • the invention provides for stable formulations, which is preferably a phosphate buffer with saline or a chosen salt, as well as preserved solutions and formulations containing a preservative as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one IL-21 agonist in a pharmaceutically acceptable formulation.
  • Preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent.
  • Any suitable concentration or mixture can be used as known in the art, such as 0.001-5%, or any range or value therein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, O.4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein.
  • Non-limiting examples include, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005- 1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.
  • 0.1-2% m-cresol e.g., 0.2, 0.3. 0.4, 0.5, 0.9
  • the invention provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of at least one IL-21 agonist with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater.
  • the invention further comprises an article of manufacture, comprising packaging material, a first vial comprising lyophilized at least one IL-21 agonist, and a second vial comprising an aqueous diluent of prescribed buffer or preservative, wherein said packaging material comprises a label that instructs a patient to reconstitute the at least one IL-21 agonist in the aqueous diluent to form a solution that can be held over a period of twenty-four hours or greater.
  • the at least one IL-21agonist used in accordance with the present invention can be produced by recombinant means, including from mammalian cell or transgenic preparations, or can be purified from other biological sources, as described herein or as known in the art.
  • the range of at least one IL-21 agonist in the product of the present invention includes amounts yielding upon reconstitution, if in a wet/dry system, concentrations from about 1.0 ⁇ g/ml to about 1000 mg/ml, although lower and higher concentrations are operable and are dependent on the intended delivery vehicle, e.g., solution formulations will differ from transdermal patch, pulmonary, transmucosal, or osmotic or micro pump methods.
  • the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative.
  • preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof.
  • concentration of preservative used in the formulation is a concentration sufficient to yield an anti-microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan.
  • excipients e.g. isotonicity agents, buffers, antioxidants, preservative enhancers
  • An isotonicity agent such as glycerin, is commonly used at known concentrations.
  • a physiologically tolerated buffer is preferably added to provide improved pH control.
  • the formulations can cover a wide range of pHs, such as from about pH 4 to about pH 10, and preferred ranges from about pH 5 to about pH 9, and a most preferred range of about 6.0 to about 8.0.
  • the formulations of the present invention have pH between about 6.8 and about 7.8.
  • Preferred buffers include phosphate buffers, most preferably sodium phosphate, particularly phosphate buffered saline (PBS).
  • polyoxyethylene polyoxypropylene block copolymers and PEG (polyethylene glycol) or non- ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyls, other block co-polymers, and chelators such as EDTA and EGTA can optionally be added to the formulations or compositions to reduce aggregation.
  • PEG polyethylene glycol
  • Pluronic® polyls polyethylene glycol
  • chelators such as EDTA and EGTA
  • the formulations of the present invention can be prepared by a process which comprises mixing at least one IL-21 agonist and a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in an aqueous diluent.
  • a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in an aqueous
  • a measured amount of at least one IL-21 agonist in buffered solution is combined with the desired preservative in a buffered solution in quantities sufficient to provide the protein and preservative at the desired concentrations.
  • Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that may be optimized for the concentration and means of administration used.
  • the claimed formulations can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one 1L-21 agonist that is reconstituted with a second vial containing water, a preservative and/or excipients, preferably a phosphate buffer and/or saline and a chosen salt, in an aqueous diluent.
  • a preservative and/or excipients preferably a phosphate buffer and/or saline and a chosen salt, in an aqueous diluent.
  • Either a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus can provide a more convenient treatment regimen than currently available.
  • the present claimed articles of manufacture are useful for administration over a period of immediately to twenty-four hours or greater. Accordingly, the presently claimed articles of manufacture offer significant advantages to the patient.
  • Formulations of the invention can optionally be safely stored at temperatures of from about 2 to about 40 0 C and retain the biologically activity of the protein for extended periods of time, thus, allowing a package label indicating that the solution can be held and/or used over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used, such label can include use up to 1-12 months, one-half, one and a half, and/or two years.
  • the solutions of at least one IL-21 agonist in the invention can be prepared by a process that comprises mixing at least one agonist in an aqueous diluent. Mixing is carried out using conventional dissolution and mixing procedures. To prepare a suitable diluent, for example, a measured amount of at least one agonist in water or buffer is combined in quantities sufficient to provide the protein and optionally a preservative or buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that may be optimized for the concentration and means of administration used.
  • the claimed products can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one IL-21 agonist that is reconstituted with a second vial containing the aqueous diluent. Either a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available.
  • the claimed products can be provided indirectly to patients by providing to pharmacies, clinics, or other such institutions and facilities, clear solutions or dual vials comprising a vial of lyophilized at least one IL-21 agonist that is reconstituted with a second vial containing the aqueous diluent.
  • the clear solution in this case can be up to one liter or even larger in size, providing a large reservoir from which smaller portions of the at least one agonist solution can be retrieved one or multiple times for transfer into smaller vials and provided by the pharmacy or clinic to their customers and/or patients.
  • Recognized devices comprising these single vial systems include those pen- injector devices for delivery of a solution such as BD Pens, BD Autojector ® , Humaject ® ' NovoPen ® , B-D ® Pen, AutoPen ® , and OptiPen ® , GenotropinPen ® , Genotronorm Pen ® , Humatro Pen ® , Reco-Pen ® , Roferon Pen ® , Biojector ® , Iject ® , J-tip Needle-Free Injector ® , Intraject ® , Medi-Ject ® , e.g., as made or developed by Becton Dickensen (Franklin Lakes, NJ, www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com; Bioject, Portland, Oregon (www.bioject.com); National Medical Products , Weston Medical (Pe
  • the products presently claimed include packaging material.
  • the packaging material provides, in addition to the information required by the regulatory agencies, the conditions under which the product can be used.
  • the packaging material of the present invention provides instructions to the patient to reconstitute the at least one IL-21 agonist in the aqueous diluent to form a solution and to use the solution over a period of 2-24 hours or greater for the two vial, wet/dry, product.
  • the label indicates that such solution can be used over a period of 2-24 hours or greater.
  • the presently claimed products are useful for human pharmaceutical product use.
  • the formulations of the present invention can be prepared by a process that comprises mixing at least one IL-21 agonist and a selected buffer, preferably a phosphate buffer containing saline or a chosen salt. Mixing the at least one agonist and buffer in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one agonist in water or buffer is combined with the desired buffering agent in water in quantities sufficient to provide the protein and buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.
  • the claimed stable or preserved formulations can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one IL-21 agonist that is reconstituted with a second vial containing a preservative or buffer and excipients in an aqueous diluent.
  • a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available
  • At least one IL-21 agonist in either the stable or preserved formulations or solutions described herein can be administered to a patient in accordance with the present invention via a variety of delivery methods including SC or EVI injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or other means appreciated by the skilled artisan, as well-known in the art.
  • the present invention also provides a method for modulating or treating at least one IL- 21 condition or pathology, in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of allergy, asthma, autoimmune diseases and the like. .
  • the present invention also provides a method for modulating or treating at least one immune related disease, in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis, gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/ admireer's granulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures, allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hyper
  • Any method of the present invention can comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one IL-21 agonist to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
  • Such a method can optionally further comprise co-administration or combination therapy for treating such immune diseases, wherein the administering of said at least one IL-21 agonist, specified portion or variant thereof, further comprises administering, before concurrently, and/or after, at least one selected from at least one multiple sclerosis therapeutic (including but not limited to, beta-interferon Ia and beta-interferon Ib (e.g., AvonexTM, RebifTM, BetaseonTM), glutiramer acetate (e.g., Copaxone), cyclophasphamide, azathioprine, glucocorticosteroids, methotrexate, Paclitaxel, 2-chlorodeoxyadenosine, mitoxantrone, IL-IO, TGBb, CD4, CD52, antegren, CDIl, CD18, TNFalpha, IL-I, IL-2, and/or CD4 antibody or antibody receptor fusion, interferon alpha, immunoglobulin, Lismide (Re
  • Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2 nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which references are entirely incorporated herein by reference.
  • TNF antagonists suitable for compositions, combination therapy, co-administration, devices and/or methods of the present invention include, but are not limited to, anti-TNF agonists, antigen-binding fragments thereof, and receptor molecules which bind specifically to TNF; compounds which prevent and/or inhibit TNF synthesis, TNF release or its action on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (e.g., pentoxifylline and rolipram), A2b adenosine receptor agonists and A2b adenosine receptor enhancers; compounds which prevent and/or inhibit TNF receptor signalling, such as mitogen activated protein (MAP) kinase inhibitors; compounds which block and/or inhibit membrane TNF cleavage, such as metalloproteinase inhibitors; compounds which block and/or inhibit TNF activity, such as angiotensin
  • MAP mitogen activated protein
  • a "tumor necrosis factor agonist,” “TNF agonist,” “TNF ⁇ agonist,” or fragment and the like decreases, blocks, inhibits, abrogates or interferes with TNF ⁇ activity in vitro, in situ and/or preferably in vivo.
  • a suitable TNF human agonist of the present invention can bind TNF ⁇ and includes anti-TNF agonists, antigen-binding fragments thereof, and specified mutants or domains thereof that bind specifically to TNF ⁇ .
  • a suitable TNF antibody or fragment can also decrease block, abrogate, interfere, prevent and/or inhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and/or synthesis.
  • Cytokines include any known cytokine. See, e.g., CopewithCytokines.com. Cytokine antagonists include, but are not limited to, any agonist, fragment or mimetic, any soluble receptor, fragment or mimetic, any small molecule antagonist, or any combination thereof.
  • Any method of the present invention can comprise a method for treating a IL-21 mediated disorder, comprising administering an effective amount of a composition or pharmaceutical composition comprising at least one IL-21 agonist to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
  • treatment of pathologic conditions is effected by administering an effective amount or dosage of at least one IL-21 Ig related protein composition that total, on average, a range from at least about 0.0001 to 500 milligrams of at least one IL-21agonist /kilogram of patient per dose, and preferably from at least about 0.001 to 100 milligrams agonist /kilogram of patient per single or multiple administration, depending upon the specific activity of contained in the composition.
  • the effective serum concentration can comprise 0.001-5000 ⁇ g/ml serum concentrations per single or multiple administrations.
  • Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved.
  • Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100 mg/kg/administration, or any range, value or fraction thereof, or to achieve
  • the dosage administered can vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • a dosage of active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight.
  • 0.1 to 50, and preferably 0.1 to 10 milligrams per kilogram per administration or in sustained release form is effective to obtain desired results.
  • treatment of humans or animals can be provided as a onetime or periodic dosage of at least one agonist of the present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively or additionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or alternatively or additionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
  • Dosage forms (composition) suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of active ingredient per unit or container. Ih these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5-99.999% by weight based on the total weight of the composition.
  • the agonist can be formulated as a solution, suspension, emulsion or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle.
  • parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives).
  • the formulation is sterilized by known or suitable techniques.
  • Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
  • IL-21 agonists of the present invention can be delivered in a carrier, as a solution, emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or other modes described here within or known in the art.
  • Formulations for parenteral administration can contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier or humidifier and a suspending agent, according to known methods.
  • Agents for injection can be a non-toxic, non-orally administrable diluting agent such as aqueous solution or a sterile injectable solution or suspension in a solvent.
  • As the usable vehicle or solvent water, Ringer's solution, isotonic saline, etc.
  • sterile involatile oil can be used as an ordinary solvent, or suspending solvent.
  • any kind of involatile oil and fatty acid can be used, including natural or synthetic or semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthetic mono- or di- or tri- glycerides.
  • Parental administration is known in the art and includes, but is not limited to, conventional means of injections, a gas pressured needle-less injection device as described in U.S. Pat. No. 5,851,198, and a laser perforator device as described in U.S. Pat. No. 5,839,446 entirely incorporated herein by reference.
  • the invention further relates to the administration of at least one IL-21 agonist by parenteral, subcutaneous, intramuscular, intravenous, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.
  • An IL-21 agonist compositions can be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms such as creams and suppositories; for buccal, or sublingual administration particularly in the form of tablets or capsules; or intranasally particularly in the form of powders, nasal drops or aerosols or certain agents; or transdermally particularly in the form of a gel, ointment, lotion, suspension or patch delivery system with chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or to increase the drug concentration in the transdermal patch (Junginger, et al.
  • At least one IL-21 agonist composition is delivered in a particle size effective for reaching the lower airways of the lung or sinuses.
  • at least one IL-21 agonist can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation. These devices capable of depositing aerosolized formulations in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Other devices suitable for directing the pulmonary or nasal administration of agonists are also known in the art. All such devices can use of formulations suitable for the administration for the dispensing of agonist in an aerosol.
  • Such aerosols can be comprised of either solutions (both aqueous and non aqueous) or solid particles.
  • Metered dose inhalers like the Ventolin ® metered dose inhaler, typically use a propellant gas and require actuation during inspiration (See, e.g., WO 94/16970, WO 98/35888).
  • Dry powder inhalers like TurbuhalerTM (Astra), Rotahaler ® (Glaxo), Diskus ® (Glaxo), SpirosTM inhaler (Dura), devices marketed by Inhale Therapeutics, and the Spinhaler ® powder inhaler (Fisons), use breath-actuation of a mixed powder (US 4668218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, US 5458135 Inhale, WO 94/06498 Fisons, entirely incorporated herein by reference).
  • Nebulizers like AERxTM Aradigm, the Ultravent ® nebulizer (Mallinckrodt), and the Acorn II ® nebulizer (Marquest Medical Products) (US 5404871 Aradigm, WO 97/22376), the above references entirely incorporated herein by reference, produce aerosols from solutions, while metered dose inhalers, dry powder inhalers, etc. generate small particle aerosols.
  • These specific examples of commercially available inhalation devices are intended to be a representative of specific devices suitable for the practice of this invention, and are not intended as limiting the scope of the invention.
  • a composition comprising at least one IL-21 agonist is delivered by a dry powder inhaler or a sprayer.
  • an inhalation device for administering at least one agonist of the present invention.
  • delivery by the inhalation device is advantageously reliable, reproducible, and accurate.
  • the inhalation device can optionally deliver small dry particles, e.g. less than about 10 ⁇ m, preferably about 1-5 ⁇ m, for good respirability.
  • a spray including IL-21 agonist composition protein can be produced by forcing a suspension or solution of at least one IL-21 agonist through a nozzle under pressure.
  • the nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size.
  • An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed.
  • particles of at least one IL-21 agonist composition protein delivered by a sprayer have a particle size less than about 10 ⁇ m, preferably in the range of about 1 ⁇ m to about 5 ⁇ m, and most preferably about 2 ⁇ m to about 3 ⁇ m.
  • Formulations of at least one IL-21 agonist composition protein suitable for use with a sprayer typically include agonist composition protein in an aqueous solution at a concentration of about 0.1 mg to about 100 mg of at least one IL-21 agonist composition protein per ml of solution or mg/gm, or any range or value therein, e.g., but not limited to, .1, .2., .3, .4, .5, .6, .7, .8, .9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml or mg/gm.
  • the formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc.
  • the formulation can also include an excipient or agent for stabilization of the agonist composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
  • Bulk proteins useful in formulating agonist composition proteins include albumin, protamine, or the like.
  • Typical carbohydrates useful in formulating agonist composition proteins include sucrose, mannitol, lactose, trehalose, glucose, or the like.
  • the agonist composition protein formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the agonist composition protein caused by atomization of the solution in forming an aerosol.
  • a surfactant which can reduce or prevent surface-induced aggregation of the agonist composition protein caused by atomization of the solution in forming an aerosol.
  • Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 14% by weight of the formulation.
  • Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as IL-21 agonists, or specified portions or variants, can also be included in the formulation.
  • Agonist composition protein can be administered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer.
  • a nebulizer such as jet nebulizer or an ultrasonic nebulizer.
  • a compressed air source is used to create a high- velocity air jet through an orifice.
  • a low-pressure region is created, which draws a solution of agonist composition protein through a capillary tube connected to a liquid reservoir.
  • the liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating the aerosol.
  • a range of configurations, flow rates, and baffle types can be employed to achieve the desired performance characteristics from a given jet nebulizer.
  • particles of agonist composition protein delivered by a nebulizer have a particle size less than about 10 ⁇ m, preferably in the range of about 1 ⁇ m to about 5 ⁇ m, and most preferably about 2 ⁇ m to about 3 ⁇ m.
  • Formulations of at least one IL-21 agonist suitable for use with a nebulizer, either jet or ultrasonic typically include a concentration of about 0.1 mg to about 100 mg of at least one IL- 21 agonist protein per ml of solution.
  • the formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc.
  • the formulation can also include an excipient or agent for stabilization of the at least one IL-21 agonist composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
  • Bulk proteins useful in formulating at least one IL-21 agonist composition proteins include albumin, protamine, or the like.
  • Typical carbohydrates useful in formulating at least one IL-21 agonist include sucrose, mannitol, lactose, trehalose, glucose, or the like.
  • the at least one IL-21 agonist formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the at least one IL-21 agonist caused by atomization of the solution in forming an aerosol.
  • a surfactant can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 4% by weight of the formulation.
  • Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan mono-oleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as agonist protein can also be included in the formulation.
  • a propellant In a metered dose inhaler (MDI), a propellant, at least one IL-21 agonist, and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol, preferably containing particles in the size range of less than about 10 ⁇ m, preferably about 1 ⁇ m to about 5 ⁇ m, and most preferably about 2 ⁇ m to about 3 ⁇ m.
  • the desired aerosol particle size can be obtained by employing a formulation of agonist composition protein produced by various methods known to those of skill in the art, including jet-milling, spray drying, critical point condensation, or the like.
  • Preferred metered dose inhalers include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant.
  • Formulations of at least one IL-21 agonist for use with a metered-dose inhaler device will generally include a finely divided powder containing at least one IL-21 agonist as a suspension in a non-aqueous medium, for example, suspended in a propellant with the aid of a surfactant.
  • the propellant can be any conventional material employed for this purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a), HFA-227 (hydrofluroalkane- 227), or the like.
  • the propellant is a hydrofluorocarbon.
  • the surfactant can be chosen to stabilize the at least one IL-21 agonist as a suspension in the propellant, to protect the active agent against chemical degradation, and the like.
  • Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid, or the like. Ih some cases solution aerosols are preferred using solvents such as ethanol. Additional agents known in the art for formulation of a protein such as protein can also be included in the formulation.
  • Formulations for oral rely on the co-administration of adjuvants (e.g., resorcinols and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to increase artificially the permeability of the intestinal walls, as well as the co-administration of enzymatic inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation.
  • adjuvants e.g., resorcinols and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether
  • enzymatic inhibitors e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol
  • the active constituent compound of the solid-type dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride.
  • at least one additive including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride.
  • These dosage forms can also contain other type(s) of additives, e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, .alpha. -tocopherol, antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening agent, flavoring agent, perfuming agent, etc.
  • inactive diluting agent e.g., lubricant such as magnesium stearate, paraben
  • preserving agent such as sorbic acid, ascorbic acid, .alpha. -tocopherol
  • antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening agent, flavoring agent, perfuming agent, etc.
  • Tablets and pills can be further processed into enteric-coated preparations.
  • the liquid preparations for oral administration include emulsion, syrup, elixir, suspension and solution preparation
  • Liposomes have also been described as drag delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carrier compounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 are used to deliver biologically active agents orally are known in the art.
  • compositions and methods of administering at least one 1L-21 agonist include an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat. Nos. 5,514,670).
  • Mucous surfaces suitable for application of the emulsions of the present invention can include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomachic, intestinal, and rectal routes of administration.
  • Formulations for vaginal or rectal administration can contain as excipients, for example, polyalkyleneglycols, vaseline, cocoa butter, and the like.
  • Formulations for intranasal administration can be solid and contain as excipients, for example, lactose or can be aqueous or oily solutions of nasal drops.
  • excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like (U.S. Pat. Nos. 5,849,695).
  • the at least one IL-21 agonist is encapsulated in a delivery device such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated).
  • a delivery device such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated).
  • suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and combinations thereof (U.S. Pat. Nos. 5,814,599).
  • a dosage form can contain a pharmaceutically acceptable non-toxic salt of the compounds that has a low degree of solubility in body fluids, for example, (a) an acid addition salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) a salt with a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, or with an organic cation formed from e.g., N,N'-dibenz
  • the compounds of the present invention or, preferably, a relatively insoluble salt such as those just described can be formulated in a gel, for example, an aluminum monostearate gel with, e.g. sesame oil, suitable for injection.
  • Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like.
  • Another type of slow release depot formulation for injection would contain the compound or salt dispersed for encapsulated in a slow degrading, non-toxic, non-antigenic polymer such as a polylactic acid/polyglycolic acid polymer for example as described in U.S. Pat. No. 3,773,919.
  • the compounds or, preferably, relatively insoluble salts such as those described above can also be formulated in cholesterol matrix silastic pellets, particularly for use in animals. Additional slow release, depot or implant formulations, e.g. gas or liquid liposomes are known in the ' literature (U.S. Pat. Nos. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems", J. R. Robinson ed., Marcel Dekker, Inc., N.Y., 1978). [238] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
  • Example 1 Generation, Cloning and Expression of an IL-21 immunoglobulin derived protein in Mammalian Cells
  • IL-21 agonists are generated using know methods, such as murine or transgenic mice expressing human antibodies that are immunized with human IL-21receptor or other suitable IL- 21 ligand, and for which B cells are isolated, cloned and selected for specificity and inhibiting activity for IL-21 (preferably with little or no inhibition of IL- 12 activity) using known methods ' and assays, e.g., as known in the art and as described herein (see, e.g., www.copewithcvtokines.de, under BL-21 and IL-12, for description and references to IL-21 proteins, IL-21 assays, entirely incorporated herein by reference, as known in the art).
  • Clones expressing IL-21 receptor specific antibodies or fusion proteins, as IL-21 agonists of the present invention are selected so that they agonize at least one IL-21 biological activity.
  • the heavy chain, light chain CDRs, variable regions, or variable and constant regions are cloned and put into appropriate expression vectors.
  • a typical mammalian expression vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the agonist coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing.
  • LTRS long terminal repeats
  • CMV cytomegalovirus
  • cellular elements can also be used (e.g., the human actin promoter).
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pIRESlneo, pRetro- Off, pRetro-On, PLXSN, or pLNCX (Clontech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
  • vectors such as pIRESlneo, pRetro- Off, pRetro-On, PLXSN, or pLNCX (Clontech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hy
  • Mammalian host cells that could be used include human HeIa 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QCl-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
  • the transfected gene can also be amplified to express large amounts of the encoded agonist.
  • the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J. 227:277- 279 (1991); Bebbington, et al., Bio/Technology 10: 169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of agonists.
  • CHO Chinese hamster ovary
  • NSO cells are often used for the production of agonists.
  • the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment of the
  • Plasmid pC4 is used for the expression of IL-21 agonist.
  • Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).
  • the plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
  • Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (e.g., alpha minus MEM, Life Technologies, Gaithersburg, MD) supplemented with the chemotherapeutic agent methotrexate.
  • a selective medium e.g., alpha minus MEM, Life Technologies, Gaithersburg, MD
  • MTX methotrexate
  • a second gene is linked to the DHFR gene, it is usually co-amplified and over-expressed. It is known in the art that this approach can be used to develop cell lines carrying more than 1,000 copies of the amplified gene(s). Subsequently, when the methotrexate is withdrawn, cell lines are obtained that contain the amplified gene integrated into one or more chromosome(s) of the host cell.
  • Plasmid ⁇ C4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438- 447 (1985)) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)). Downstream of the promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene.
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • high efficiency promoters can also be used for the expression, e.g., the human b-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
  • Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the IL-21 in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).
  • Other signals e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the ' beginning, e.g., G418 plus methotrexate.
  • the plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated from a 1% agarose gel.
  • the DNA sequence encoding the complete IL-21 agonist is used, corresponding to HC and LC variable regions of an IL-21 agonist of the present invention, according to known method steps.
  • Isolated nucleic acid encoding a suitable human constant region i.e., HC and LC regions
  • HC and LC regions are also used in this construct (e.g., as provided in vector pl351).
  • variable and constant region encoding DNA and the dephosphorylated vector are then ligated with T4 DNA ligase.
  • E. coli HBlOl or XL-I Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
  • CHO cells lacking an active DHPR gene are used for transfection. 5 ⁇ g of the expression plasmid ⁇ C4 is cotransfected with 0.5 ⁇ g of the plasmid pSV2-neo using lipofectin.
  • the plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
  • the cells are seeded in alpha minus MEM supplemented with 1 ⁇ g /ml G418.
  • the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 ⁇ g /ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well Petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).
  • Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained that grow at a concentration of 100 - 200 mM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.
  • EXAMPLE 2 IL-21 and IL-21 Agonists inhibit IgE Isotype Switch and IgE production in mouse allergic model.
  • mice genetically deficient in interleukin-21 were generated by gene targeting.
  • IL-21 " ' " mice display reductions in the numbers of splenic, thymic, peripheral blood, and lymph node (LN) CD8 T cells, NK cells and NK T cells, and increases in the numbers of granulocytes.
  • Spleen B cells in IL-21 7" mice exhibited a slightly decreased response to anti- CD40 stimulation.
  • IgE levels are remarkably higher in IL ⁇ l " ' ' mice.
  • IL-21 '1' mice also displayed an enhanced IL- 15 mediated resting NK cell expansion, which suggests that IL-21 inhibits IL- 15 mediated resting NK cell expansion.
  • IL-21 " ' ' mice exhibited a decreased response to anti-CD3 and anti-CD28 stimulation. Since IL-21 limits resting NK expansion while promoting antigen specific T cell-mediated immunity, these data suggest that IL-21 may be a key element in the transition between innate and adaptive immune response.
  • CHS contact hypersensitivity response
  • draining LN T cell especially CD8 cell proliferation was inhibited in EL-21 " ' " mice. Consistent with the results from proliferation assays, draining lymph nodes T cells in IL-21 " ' " mice produce less cytokines including IL-4, IL-5, IL-6, TNF- ⁇ and IL-IO.
  • FITC induced-CHS response is a Th-2 driven CHS response.
  • FITC-induced CHS response was significantly increased in JL-21 '1' mice. The enhanced response was manifested as a markedly increased infiltration of inflammatory cells, increased ear thickness and elevated serum IgE levels in IL-
  • Th2 cytokines The levels of Th2 cytokines, pro-inflammatory cytokines, and chemokines were significantly higher in IL-21 "7' mice. In addition, T cell proliferation was significantly increased in response to anti-CD3 treatment. Draining lymph nodes (LN) T cells in IL-21 "7" mice produced less ThI cytokine, IFN- ⁇ , and much higher Th2 cytokines such as IL-4, IL-5, IL-13, pro- inflammatory cytokine, IL-Ib, and chemokine, MCP-I. Since oxazolone induced-CHS is predominantly a ThI dependent response and FITC-induced CHS is Th2-driven, our findings indicate that IL-21 plays an important role in modulating both ThI and Th2 immune responses.
  • IL-21 may be a key facilitator in the transition between innate and adaptive immune response.
  • IL-21 itself or its agonist may also be useful for the treatment of asthmatic and allergic diseases, infectious diseases, and cancer.
  • IL-21 genetically deficient mice were generated by homologous recombination in embryonic stem (ES) cells.
  • a gene-targeting vector was constructed in which IL-21 exons 1 and 2 were replaced with IRES-LacZ/M Clneo cassette.
  • 129sv-derived ES cells were electroporated with the EL-21 targeting vector and selected in G418.
  • ES cell clones carrying a targeted IL-21 gene were identified by genomic Southern blot analysis.
  • Targeted ES were microinjected into C57BL/6. Resulting male chimeras were bred with C57BL/6 female mice.
  • the offspring were analyzed for germ-line transmission of the mutant IL-21 allele by PCR and Southern blot analyses. Mice heterozygous for IL-21 mutant were intercrossed to generate IL ⁇ l ⁇ mice.
  • PCR primers were designed using Vector NTI 7.1 (InfoMax, Inc. Bethesda, MD) and synthesized by MWG (High Point, NC). Sequences of the primer set are listed below, mkl-ss: 5'-TGCCGAAATGGTCCATCAAA-S'; mkl-as: 5'-TGCGGTAGTTCA- GGCAGTTC-3'; mwl-ss:5'-CATGGAGAGGACCCTTGTCTG-3'; mwl-as: 5'-TCTTACCTTT- ACATCTTGTGGAGCT-3'.
  • Mouse tails (1-2 mm) were incubated with 0.2 ml NID-buffer (50 mM KCl, 10 mM Tris/Cl pH8.3,2 mM MgC12, 0.1 mg/mL gelatin, 0.45% NP40, 0.45% Tween )(Sigma, St. Louis, MT) and 0.7 ⁇ L of 20 mg/mL of proteinase K (Promega, Madison, Wl) at 56°C overnight. Proteinase K was inactivated by incubation of the sample at 95°C for 10 min and supernatants were spun down for PCR.
  • NID-buffer 50 mM KCl, 10 mM Tris/Cl pH8.3,2 mM MgC12, 0.1 mg/mL gelatin, 0.45% NP40, 0.45% Tween
  • Proteinase K was inactivated by incubation of the sample at 95°C for 10 min and supernatants were spun down for PCR.
  • PCR reaction was performed in 50 ⁇ L volume containing 5 ⁇ L of tail lysate, Ix PCR buffer, 0.8mM dNTP (Applied Biosystem, Forster City, CA), 0.2 ⁇ M of each of PCR primers (mwl-ss, mwl-as, mkl-ss, mkl-as), 0.5 ⁇ L of Taq polymerase (Promega, Madison, WI) and water. The reaction was then initiated at 95°C for 5 minutes followed by 30 cycles of 95°C for 30 seconds; 54°C for 30 seconds; and 72°C for 1 minute. A multiplex PCR screening of mouse tail lysates was developed.
  • Two primer pairs (mwl-ss, mwl-as, mkl-ss, mkl-as) were added to one PCR reaction and tail lysate was used as a template. This method can distinguish between the wild type (WT) and the targeted alleles. Primers mwl-ss and mwl-as amplify a 298 bp product from the wild type allele. Primers mkl and mkl-as amplify a 516 bp product from the targeted allele ( ⁇ -21 " ' " ).
  • T-cell and B-cell proliferation Primary cells were plated 100 ⁇ L/well at a density of 5X105 cells per ml in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (JRH Bioscience, Lenexa, KS), 1 mM sodium pyruvate (JRH Bioscience), 2 mM L- glutamine (Invitrogen), 0.1 mM MEM non-essential amino acids (JRH Bioscience).
  • NK cell expansion Spleen cells were incubated in the same medium as described above with 10 ng/ml of IL-15 with and without 100 ng/ml of mIL-21 for 7 days. At the end of 7 days, cell surface markers, CD3, NKl.1 were analyzed by flow cytometry.
  • Measurement of serum IgE Blood was collected by cardiac puncture and serum harvested from each blood sample as needed for IgE measurement. Serum IgE was measured by sandwich ELISA kit (BD Bioscience, San Jose, CA).
  • IL-2r A mice appear to be normal and fertile. Blood counts are within the normal range except that the ⁇ L-21 ' ' ' mice have a higher count and percentage of granulocytes. Total body weight, and the absolute and relative lymphoid organ weights show no marked differences among different genotypes. Cell numbers of spleen and thymus are within the normal ranges among different genotypes. A slight decrease was observed in the average cell number of the peripheral LNs in the IL-21 ' ' " mice.
  • mice used throughout these studies were bred to a C57BL/6 genetic background for three generations, this may explain why only a certain proportion of the cell population showed altered phenotypes in the IL-2r /" mice.
  • IgE levels from serum samples were measured by ELISA (BD PharMingen, San Diego, CA), as shown in Figure 9, IgE levels in the IL-21 ' ' " mice were dramatically elevated.
  • CHS Contact hypersensitivity
  • mice were sensitized and challenged to elicit CHS responses to oxazolone and to FITC as described below.
  • C57BL/6 wild type and IL-21 " ' " mice were sensitized with 20 ⁇ L of 1.6% (w/v) oxazolone solution (Sigma, St Louis, MO) in ethanol on both the inside and outside of the left ear on day 1.
  • oxazolone solution Sigma, St Louis, MO
  • mice were sensitized with 20 ⁇ L of a 1% (w/v) solution of FTTC (Sigma) in 50:50 (vol/vol) acetone dibutylphtalate (DBT) on day 1, the mice were challenged on both sides of the left ear with 20 ⁇ l of a 0.5% (w/v) solution of FTTC in 50:50 DBT on day 8. Ear thickness was measured on day 1, and Ohrs, 6hrs, 24hrs, 48hrs, and 72hrs after challenge with a thickness gauge, model No 313- 196 (Dyer company, Lancaster, PA) and tissue samples collected at various time points after challenge. In the experiment of repeated oxazolone challenge, the mice were challenged with
  • Anti-mIL-21 mAb inhibits oxazolone induced CHS response.
  • BALB/c mice were injected i.p. with different doses of an anti-mIL-21 mAb, PHD131, generated by Centocor
  • mice were sensitized with 20 ⁇ L of 1.6% (w/v) oxazolone solution on day 1.
  • mice were again injected i.p. with different doses of anti-mIL-21 mAb or isotype control mAb.
  • Animals were challenged on day 8 with 20 ⁇ L of 0.8% oxazolone solution. Ear thickness was measured on day 0 and at Ohr, 8hrs, 24hrs, 48hrs, and 72hrs after challenge and tissue samples were collected at various time points after challenge.
  • 5xlO 5 cells were cultured in 200 ⁇ L of RPMI medium 1640 (Life Technologies) with 2 mM 1-glutamine, 10% FBS, 1% sodium pyruvate, 1% non essential amino-acid, 100U/ml of penicillin, and 100 ⁇ g/ml of streptomycin (Life Technologies) in response to 1 ⁇ g/ml of anti-murine CD3 monoclonal antibody (clone 145-2C11) or 1 ⁇ g/ml of anti-murine CD28 monoclonal antibody (BD Bioscience) for a period of 72 hr. 100 ⁇ L of supernatant was taken for cytokine detection (Linco research, Inc. St. Charles, MO). Per kit directions (Packard), the cells were then lysed and ATP-lite substrate was added. Luminescence was measured using a Topcount plate reader (Packard).
  • mice The level of IFN-D in ⁇ -,-21 " ' " mice was significantly lower than that in wild type mice 24 hrs post-challenge ( Figure 12).
  • the other cytokines such as IL-4, IL-5, TNF-oc, IL-l ⁇ , IL-12, IL-2, IL-10 and GMCSF in ears were not altered in JL-21 '1' mice.
  • draining LN T cell proliferation and cytokine production were assessed. T cell proliferation was significantly inhibited in ⁇ -,-21 ' ' " mice.
  • FITC-indueed CHS is Th2-driven.
  • FITC-induced CHS response was increased in TL-2l ⁇ ' ⁇ mice.
  • the ear thickness was dramatically increased in TL-2V J ⁇ mice when compared to the response in wild type mice ( Figure 17).
  • the enhanced response wasmanifested as a markedly increased infiltration of inflammatory cells, increased ear thickness in JL-21 '1" mice ( Figure 18).
  • the CHS response reached a maximum at 24 hr followed by a decrease after 48-72 hr.
  • IgE level is thought to be a reliable marker of Th2 activity, we measured total serum IgE level 72 hr after FlTC challenge.
  • mice The IgE level of FITC challenged IL-21 ' ' " mice was higher (4.311 ng/ml) compared to non-challenged IL-2r /" mice (52 ng/ml) or FITC challenged WT (1168 ng/ml) as shown in Figure 19. Since eosinophils are typically present in Th2- mediated allergic diseases, we next measured eotaxin level as a specific index of eosinophil infiltration into the challenged skin. Eotaxin level in ear samples taken from FITC challenged IL-21 "7' mice was significantly increased compared to that in WT ( Figure 20). These data clearly indicate that more eosinophils migrate into the skin of FITC challenged IL-2r ;" mice.
  • cytokines and chemokines were examined.
  • the levels of Th2 cytokines, pro-inflammatory cytokines, and chemokines were significantly higher in IL-2r A mice as shown in Figure 21.
  • T cell proliferation was significantly increased in response to anti-CD3 treatment (Figure 22). Draining lymph nodes T cells in IL-21 "7" mice produced less ThI cytokine, IFN-g, and much higher Th2 cytokines such as IL-4, IL-5, IL- 13 pro-inflammatory cytokine, IL-Ib, and chemokine, MCP-I ( Figure 23).
  • IL-21 limits NK cell responses and promotes antigen- specific T cell activation: a mediator of the transition from innate to adaptive immunity.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des méthodes de traitement d'au moins un état ou une pathologie lié à l'IL-21, qui incluent des procédés, des dispositifs et des compositions thérapeutiques. Ladite pathologie implique l'augmentation de la production d'IgE ou la commutation isotopique d'IgE.
PCT/US2006/012749 2005-03-31 2006-03-31 Methodes et compositions de traitement de pathologies afferentes a l'il-21 Ceased WO2006105538A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US66678405P 2005-03-31 2005-03-31
US60/666,784 2005-03-31

Publications (2)

Publication Number Publication Date
WO2006105538A2 true WO2006105538A2 (fr) 2006-10-05
WO2006105538A3 WO2006105538A3 (fr) 2009-04-23

Family

ID=37054232

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/012749 Ceased WO2006105538A2 (fr) 2005-03-31 2006-03-31 Methodes et compositions de traitement de pathologies afferentes a l'il-21

Country Status (1)

Country Link
WO (1) WO2006105538A2 (fr)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009047360A1 (fr) * 2007-10-11 2009-04-16 Novo Nordisk A/S Anticorps il-21
WO2010055366A3 (fr) * 2007-12-07 2010-10-21 Zymogenetics, Inc. Anticorps monoclonaux anti-il-21 humain
US8455449B2 (en) 2011-01-18 2013-06-04 Bioniz, Llc Compositions and methods for modulating γ-c-cytokine activity
US8592462B2 (en) 2008-11-10 2013-11-26 Intermune, Inc. Pirfenidone treatment for patients with atypical liver function
WO2014205501A1 (fr) 2013-06-27 2014-12-31 Monash University Protéines de liaison il-21 et leurs utilisations
WO2016094962A1 (fr) 2014-12-19 2016-06-23 Monash University Anticorps il -21
US9388241B2 (en) 2005-11-28 2016-07-12 Zymogenetics, Inc. Anti-human IL-21 antibodies
US9959384B2 (en) 2013-12-10 2018-05-01 Bioniz, Llc Methods of developing selective peptide antagonists
US10030059B2 (en) 2015-10-09 2018-07-24 Bioniz, Llc Modulators of gamma-C-cytokine activity
US10238625B2 (en) 2015-08-07 2019-03-26 Respivant Sciences Gmbh Methods for the treatment of mast cell related disorders with mast cell stabilizers
US10238628B2 (en) 2014-02-10 2019-03-26 Respivant Sciences Gmbh Mast cell stabilizers treatment for systemic disorders
US10265296B2 (en) 2015-08-07 2019-04-23 Respivant Sciences Gmbh Methods for the treatment of systemic disorders treatable with mast cell stabilizers, including mast cell related disorders
US10265267B2 (en) 2016-08-31 2019-04-23 Respivant Sciences Gmbh Cromolyn compositions for treatment of chronic cough due to idiopathic pulmonary fibrosis
US10561635B2 (en) 2016-10-07 2020-02-18 Respivant Sciences Gmbh Cromolyn compositions for treatment of pulmonary fibrosis
US10835512B2 (en) 2014-02-10 2020-11-17 Respivant Sciences Gmbh Methods of treating respiratory syncytial virus infections
US12030936B2 (en) 2019-05-03 2024-07-09 Bioniz Therapeutics, Inc. Modulating the effects of gamma-c-cytokine signaling for the treatment of alopecia and alopecia associated disorders

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CAVEN ET AL.: 'rHull-21 Enhances IgE Production in Human PBMC and Purified Tonsilar B Cellls Stimulated with IL-4 and alphaCD40 in a Cell Number, IL Concentration, and Proliferation Dependent Manner.' FASEB JOURNAL vol. 18, no. 4-5, 2004, page A1125 *
OZAKI ET AL.: 'A Critical Role for IL-21 in Regulating Immunoglobulin Production.' SCIENCE. vol. 298, November 2002, pages 1630 - 1634 *
SHANG ET AL.: 'IL-2 but not IFN-g production in a mouse model of allergy.' FASEB JOURNAL. vol. 19, no. 4, March 2005, page A939 *
SUTO ET AL.: 'Interleukin 21 prevents antigen-induced IgE production by inhibiting germ line Ce ranscription of IL-4-stimulated B cells.' BLOOD. vol. 100, no. 13, December 2002, pages 4565 - 4573 *
WOOD ET AL.: 'IL-21 efects on human IgE production in response to IL-4 or IL-13.' CELLULAR IMMUNOLOGY. vol. 231, 2004, pages 133 - 145 *

Cited By (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9388241B2 (en) 2005-11-28 2016-07-12 Zymogenetics, Inc. Anti-human IL-21 antibodies
WO2009047360A1 (fr) * 2007-10-11 2009-04-16 Novo Nordisk A/S Anticorps il-21
US8361470B2 (en) 2007-12-07 2013-01-29 Zymogenetics, Inc. Methods of treatment using anti-human IL-21 monoclonal antibodies
EP2426146A3 (fr) * 2007-12-07 2012-05-30 ZymoGenetics, Inc. Anticorps monoclonaux IL-21 anti-humains
US8226948B1 (en) 2007-12-07 2012-07-24 Zymogenetics, Inc. Anti-human IL-21 monoclonal antibodies
US8241629B2 (en) 2007-12-07 2012-08-14 Zymogenetics, Inc. Anti-human IL-21 monoclonal antibodies
KR101591316B1 (ko) 2007-12-07 2016-02-05 지모제넥틱스, 인코포레이티드 항-사람 ｉｌ-21 단클론성 항체
CN102027011A (zh) * 2007-12-07 2011-04-20 津莫吉尼蒂克斯公司 抗人il-21单克隆抗体
CN103772502A (zh) * 2007-12-07 2014-05-07 津莫吉尼蒂克斯公司 抗人il-21单克隆抗体
CN102027011B (zh) * 2007-12-07 2014-09-03 津莫吉尼蒂克斯公司 抗人il-21单克隆抗体
EP3103813A1 (fr) * 2007-12-07 2016-12-14 ZymoGenetics, Inc. Anticorps monoclonaux il-21 anti-humains
WO2010055366A3 (fr) * 2007-12-07 2010-10-21 Zymogenetics, Inc. Anticorps monoclonaux anti-il-21 humain
US8592462B2 (en) 2008-11-10 2013-11-26 Intermune, Inc. Pirfenidone treatment for patients with atypical liver function
US8609701B2 (en) 2008-11-10 2013-12-17 Intermune, Inc. Pirfenidone treatment for patients with atypical liver function
US9675672B2 (en) 2011-01-18 2017-06-13 Bioniz, Llc Compositions and methods for modulating gamma-C-cytokine activity
US9951105B2 (en) 2011-01-18 2018-04-24 Bioniz, Llc Methods of developing selective peptide antagonists
US10808009B2 (en) 2011-01-18 2020-10-20 Bioniz, Llc Peptide conjugates
US9133243B2 (en) 2011-01-18 2015-09-15 Bioniz, Llc Compositions and methods for modulating γ-c-cytokine activity
US9133244B2 (en) 2011-01-18 2015-09-15 Bioniz, Llc Compositions and methods for modulating gamma-c-cytokine activity
US11708392B2 (en) 2011-01-18 2023-07-25 Bioniz, Llc Peptide conjugates
US10227382B2 (en) 2011-01-18 2019-03-12 Bioniz, Llc Method of designing a peptide and/or peptide derivative for modulating gamma-c-cytokine activity
US11834519B2 (en) 2011-01-18 2023-12-05 Bioniz Therapeutics, Inc. Compositions and methods for modulating γ-c-cytokine activity
US8455449B2 (en) 2011-01-18 2013-06-04 Bioniz, Llc Compositions and methods for modulating γ-c-cytokine activity
AU2014302028B2 (en) * 2013-06-27 2019-09-19 Monash University IL-21 binding proteins and uses thereof
CN105579470A (zh) * 2013-06-27 2016-05-11 莫纳什大学 Il-21结合蛋白和其用途
CN105579470B (zh) * 2013-06-27 2021-05-25 莫纳什大学 Il-21结合蛋白和其用途
JP2016529213A (ja) * 2013-06-27 2016-09-23 モナッシュ ユニバーシティ Ｉｌ−２１結合タンパク質及びその使用
EP3013858A4 (fr) * 2013-06-27 2017-01-25 Monash University Protéines de liaison il-21 et leurs utilisations
US10077301B2 (en) 2013-06-27 2018-09-18 Monash University IL-21 binding proteins
WO2014205501A1 (fr) 2013-06-27 2014-12-31 Monash University Protéines de liaison il-21 et leurs utilisations
US10787510B2 (en) 2013-06-27 2020-09-29 Monash University Methods of treating cancer using an IL-21 agonist
US11462297B2 (en) 2013-12-10 2022-10-04 Bioniz, Llc Selective peptide antagonists
US9959384B2 (en) 2013-12-10 2018-05-01 Bioniz, Llc Methods of developing selective peptide antagonists
US10854312B2 (en) 2013-12-10 2020-12-01 Bioniz, Llc Selective peptide antagonists
US10238628B2 (en) 2014-02-10 2019-03-26 Respivant Sciences Gmbh Mast cell stabilizers treatment for systemic disorders
US10835512B2 (en) 2014-02-10 2020-11-17 Respivant Sciences Gmbh Methods of treating respiratory syncytial virus infections
US10398673B2 (en) 2014-02-10 2019-09-03 Respivant Services GmbH Mast cell stabilizers treatment for systemic disorders
US10940212B2 (en) 2014-12-19 2021-03-09 Monash University IL-21 agonist antibodies and methods of treatment using same
WO2016094962A1 (fr) 2014-12-19 2016-06-23 Monash University Anticorps il -21
AU2015367224B2 (en) * 2014-12-19 2020-12-10 Monash University IL-21 antibodies
EP3233120A4 (fr) * 2014-12-19 2018-05-30 Monash University Anticorps il -21
US10391078B2 (en) 2015-08-07 2019-08-27 Respivant Sciences Gmbh Methods for the treatment of mast cell related disorders with mast cell stabilizers
US10596146B2 (en) 2015-08-07 2020-03-24 Respivant Sciences Gmbh Methods for the treatment of systemic disorders treatable with mast cell stabilizers, including mast cell related disorders
US10265296B2 (en) 2015-08-07 2019-04-23 Respivant Sciences Gmbh Methods for the treatment of systemic disorders treatable with mast cell stabilizers, including mast cell related disorders
US10238625B2 (en) 2015-08-07 2019-03-26 Respivant Sciences Gmbh Methods for the treatment of mast cell related disorders with mast cell stabilizers
US10030059B2 (en) 2015-10-09 2018-07-24 Bioniz, Llc Modulators of gamma-C-cytokine activity
US11400134B2 (en) 2015-10-09 2022-08-02 Bioniz, Llc Modulating gamma-c-cytokine activity
US10030058B2 (en) 2015-10-09 2018-07-24 Bioniz, Llc Modulating gamma-C-cytokine activity
US10463613B2 (en) 2016-08-31 2019-11-05 Respivant Sciences Gmbh Cromolyn compositions for treatment of chronic cough due to idiopathic pulmonary fibrosis
US10265267B2 (en) 2016-08-31 2019-04-23 Respivant Sciences Gmbh Cromolyn compositions for treatment of chronic cough due to idiopathic pulmonary fibrosis
US10583113B2 (en) 2016-10-07 2020-03-10 Respivant Sciences Gmbh Cromolyn compositions for treatment of pulmonary fibrosis
US10561635B2 (en) 2016-10-07 2020-02-18 Respivant Sciences Gmbh Cromolyn compositions for treatment of pulmonary fibrosis
US12030936B2 (en) 2019-05-03 2024-07-09 Bioniz Therapeutics, Inc. Modulating the effects of gamma-c-cytokine signaling for the treatment of alopecia and alopecia associated disorders

Also Published As

Publication number Publication date
WO2006105538A3 (fr) 2009-04-23

Similar Documents

Publication Publication Date Title
US7807471B2 (en) IL-23p40 specific immunoglobulin derived proteins, compositions, epitopes, methods and uses
AU2004239288B2 (en) IL-23p40 specific immunoglobulin derived proteins, compositions, methods and uses
CA2418961C (fr) Anticorps, compositions, procedes anti-il-12 et leur utilisation
US20060246075A1 (en) Anti-amyloid antibodies, compositions, methods and uses
US20030157105A1 (en) Anti-p40 immunglobulin derived proteins, compositions, methods and uses
US20050266005A1 (en) Methods and compositions for treating IL-3 related pathologies
EP1644416A2 (fr) Proteines derivees de l'immunoglobuline anti-cible et genetiquement modifiees, compositions, procedes et utilisations
AU2003243785A1 (en) Cngh0004 polypeptides, antibodies, compositions, methods and uses
AU2004274390A1 (en) Anti-amyloid antibodies, compositions, methods and uses
AU2008312611A1 (en) Human anti-amyloid antibodies, compositions, methods and uses
WO2006105538A2 (fr) Methodes et compositions de traitement de pathologies afferentes a l'il-21
US20050232923A1 (en) Methods and compositions for treating MCP-1 related pathologies
US20190345245A1 (en) Methods of Treating Crohn's Disease with Anti-IL23 Specific Antibody
US20070160606A1 (en) Treating renal cell carcinoma with an anti-TNF human antibody or fragment
US20060246070A1 (en) Methods and compositions for treating renal cell carcinoma related pathologies
WO2003082206A2 (fr) Proteines derivees de l'immunoglobuline liees a la sclerose en plaques, compositions, methodes et utilisations relatives
US20040018195A1 (en) Diabetes-related immunoglobulin derived proteins, compositions, methods and uses
US20050266004A1 (en) Anti-human lymphotoxin alpha antibodies, compositions, methods and uses
WO2002074916A2 (fr) Proteines derivees de l'immunoglobuline de type reg, compositions, et methodes d'utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase in:

Ref country code: DE

NENP Non-entry into the national phase in:

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 06749378

Country of ref document: EP

Kind code of ref document: A2