Il-21 antagonists

a technology of il-21 and antagonists, which is applied in the field of il-21 antagonists, can solve the problems of loss of tolerance and the serious clinical consequences of autoimmunity

Inactive Publication Date: 2007-05-31
ZYMOGENETICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0005] In one aspect, the present invention provides an anti-IL-21 monoclonal antibody that binds to an antigen region of human IL-21. In certain embodiments, the monoclonal antibody binds to an antigenic region of IL-21 that is shown in SEQ ID NO: 6 from amino acid residues 97-122. In another embodiment, the monoclonal antibody binds an antigenic region as shown in SEQ ID NO: 6 from amino acid residues 145 to 148. In another embodiment, the monoclonal antibody binds an antigenic region as shown in SEQ ID NO: 6 from amino acid residues 154 to 162. In another embodiment, the monoclonal antibody binds an ant

Problems solved by technology

Sometimes, the immune system loses the ability to recognize “self” as normal and the subsequent response directed against the tissue or cells, results in loss of tolerance, a state of

Method used

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Examples

Experimental program
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Effect test

example 1

Preparation of IL-21 Proteins

[0139] IL-21 protein was produced as described in U.S. Patent Application No. 2006-0134754 and WO 04 / 055168, incorporated in its entirety herein. Briefly, a IL-21 nucleotide sequence was optimized and inserted in an E. coli expression vector which was deposited as ATCC Accession No. PTA-4853. The expression vector was introduced into E. coli strain W3110 (ATCC Accession No. 27325).

[0140] Host cells were fermented by growing E. coli strains expressing IL-21 in a suitable medium in shake flask culture to in a suitable medium and may be supplemented with carbohydrates, such as fructose, glucose, galactose, lactose, and glycerol. Isopropyl thiogalactopyranoside (IPTG) is may be added to the culture to a concentration 0.1 to 2.0 mM.

[0141] Following fermentation the cells were harvested by centrifugation, re-suspended in homogenization buffer and homogenized. After the homogenate was collected, it was resuspended a guanidine containing solution and the sup...

example 2

Preparation of IL-21 Receptor Proteins

[0143] The IL-21 receptor (also designated as zalpha11 or IL-21r) heterodimer protein can be produced as described in U.S. Patent Application No. 2002-0137677, incorporated in its entirety herein. Briefly, a vector expressing a secreted human hzalpha11 / hIL2Rgamma heterodimer is constructed. In this construct, the extracellular domain of hzalpha11 is fused to the CH1 domain of IgG γ1. The CH1 domain is cloned into a mammalian expression vector. The CL1 domain of the human κ light chain is cloned in a mammalian expression vector.

[0144] A construct having human zalpha11 fused to CH1 is made, and the vector is sequenced to confirm that the fusion is correct. A separate construct having hIL2Rgamma fused to CL1 can be also constructed. The resulting vector is sequenced to confirm that the human IL-2Rgamma / CL1 fusion is correct.

[0145] The human zalpha11 (IL-21r) and human IL-2Rgamma receptor fusions are co-expressed. Each expression vector is co-tr...

example 3

Preparation of IL-21 Monoclonal Antibodies

[0149] Rat monoclonal antibodies are prepared by immunizing 4 female Sprague-Dawley Rats (Charles River Laboratories, Wilmington, Mass.), with the purified recombinant IL-21 protein. The rats are each given an initial intraperitoneal (IP) injection of 25 μg of the purified recombinant protein in Complete Freund's Adjuvant (Pierce, Rockford, Ill.) followed by booster IP injections of 10 μg of the purified recombinant protein in Incomplete Freund's Adjuvant every two weeks. Seven days after the administration of the second booster injection, the animals are bled and serum is collected.

[0150] The IL-21-specific rat sera samples are characterized by ELISA using 1 ug / ml of the purified recombinant IL-21 receptor protein as the specific antibody target. ELISAs comprise preparing IL-21 antigen, coating the wells of a 96-well microtiter plate with the antigen, adding the rat sera of interest to the wells and incubating for a period of time to all...

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Abstract

Monoclonal antibodies are identified that bind the IL-21 protein. These antibodies are used to identify regions of the IL-21 protein to where binding neutralizes IL-21 activity. Hybridomas and methods of producing anti-IL-21 monoclonal antibodies are described. The monoclonal antibodies are useful in treating IL-21-mediated diseases, which may include autoimmune and inflammatory diseases such as pancreatitis, type I diabetes (IDDM), Graves Disease, inflammatory bowel disease (IBD), Crohn's Disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, rheumatoid arthritis, diverticulosis, systemic lupus erythematosus, psoriasis, ankylosing spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, graft vs. host disease (GVHD), cutaneous T cell lymphoma (CTCL), Sjogren's syndrome, glomerulonephritis, IgA nephropathy, graft versous host disease, transplant rejection, atopic dermatitis, anti-phospholipid syndrome, and asthma, and other autoimmune diseases.

Description

REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application Ser. No. 60 / 740,154, filed Nov. 28, 2005, which is herein incorporated by reference.BACKGROUND OF THE INVENTION [0002] The immune system is the body's primary defense against diseases caused by pathogens, namely bacteria, viruses, fungi etc, as well as against diseases caused by abnormal growth of the body's own cells and tissues (i.e. cancerous tumors). Normally, the immune system is able to distinguish between the body's normal cells or “self” and foreign pathogens or abnormal cells or “non-self”. The processes by which the immune system refrains from reacting to one's own body is called tolerance. Sometimes, the immune system loses the ability to recognize “self” as normal and the subsequent response directed against the tissue or cells, results in loss of tolerance, a state of autoimmunity. The pathologies resulting from autoimmunity often have serious clinical consequenc...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/24C12P21/08C12N5/06
CPCA61K2039/505C07K16/244Y10S530/809C07K2319/30C07K2317/34C07K2316/96A61P1/00A61P1/04A61P1/18A61P3/10A61P5/14A61P11/06A61P13/12A61P17/00A61P17/04A61P17/06A61P19/02A61P19/08A61P21/02A61P25/00A61P29/00A61P35/00A61P35/02A61P37/02A61P37/06A61P37/08A61P43/00C07K2317/76A61K39/3955C07K2317/33
Inventor SIVAKUMAR, PALLAVUR V.JASPERS, STEPHEN R.
Owner ZYMOGENETICS INC
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