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Antibodies that specifically bind to human il-15 and uses thereof

A specific and antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, specific peptides, etc.

Pending Publication Date: 2019-09-13
赛福伦有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, no therapeutic agent has been shown to be effective in the treatment of celiac disease as measured by the aforementioned endpoints

Method used

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  • Antibodies that specifically bind to human il-15 and uses thereof
  • Antibodies that specifically bind to human il-15 and uses thereof
  • Antibodies that specifically bind to human il-15 and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Transgenic rat production, immunization and hybridoma production

[0145] 1.1 IL-15 protein and IL-15Rα protein

[0146] Human interleukin 15 (IL-15) was purchased (Sigma), or soluble IL-15 receptor alpha (IL-15Rα ) (SEQ ID NO:512) was produced in the mammalian HEK293F expression system.

[0147] 1.2 Production of transgenic rats

[0148] Transgenic rats were generated as described in PCT Publication No. WO 08 / 151081. Briefly, the meganuclease expression construct is integrated into the genome of the test animal. Expression of meganucleases in germ cells causes double-strand breaks in endogenous rat immunoglobulin genes. Mating of such transgenic rats produces offspring with mutated / inactivated endogenous rat immunoglobulin genes.

[0149] Transgenic rats are further modified to carry artificial immunoglobulin genes, which allow the rats to produce antibodies with fully human variable regions.

[0150] 1.3 Immunity

[0151] To generate fully human monoclona...

Embodiment 2

[0156] Hybridoma Screening

[0157] 2.1 Selection of antibodies that bind IL-15 complexed but not uncomplexed IL-15 receptor alpha using ELISA

[0158]Microtiter plates were coated with purified IL-15 or purified IL-15Rα or purified IL-15 complexes. Briefly, microtiter plates are coated with purified protein in PBS and then blocked with an unrelated protein such as bovine serum albumin (BSA) diluted in PBS. Dilutions of hybridoma supernatants were added to each well and incubated at 37°C for 1-2 hours. Use the board 20 washes followed by incubation for 1 hour at 37°C with goat anti-human IgG Fc-specific polyclonal reagent conjugated to a suitable detection reagent (eg horseradish peroxidase) alkaline phosphatase. After washing, the plate is developed with an appropriate substrate (eg 3,3',5,5'-tetramethylbenzidine TMD) and analyzed at an OD of 405. Hybridomas producing antibodies positively reactive with the IL-15 complex but not with IL-15Ra were selected for further c...

Embodiment 3

[0165] Identify antibody candidates for further development

[0166] 3.1 Assay based on CTLL-2 cells

[0167] 1500 hybridoma samples that bound the IL-15 complex but not IL-15α were tested in a murine CTLL-2 cell-based assay to determine which neutralized the biological activity of IL-15. The CTLL-2 cell line is derived from cytotoxic T-cell lymphoma (ATCC: TIB-214) and responds to both IL-2 and IL-15.

[0168] Hybridoma supernatants (unpurified antibodies) were tested for their ability to neutralize IL-15-induced proliferation of CTLL-2 cells.

[0169] CTLL-2 cells were incubated in complete medium without IL-2 or IL-15 for 4 hours prior to testing. CTLL-2 cells (5x10 4 cells / well) were incubated with 200 pM IL-15 and IL-15 receptor α complex in a 96-well plate to induce cell proliferation. Hybridoma supernatants were added to the plates and incubated for 48 hours. Then use according to the manufacturer's instructions Inhibition of cell proliferation was assessed by ...

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Abstract

Recombinant antibodies that specifically bind to IL-15 as well as a complex of IL-15 and the IL-15 Receptor-alpha are provided. The antibodies inhibit immune cell proliferation, and are capable of usein the treatment of any autoimmune or inflammatory disease or condition where IL-15 is dysregulated, including Celiac disease.

Description

[0001] References to Sequence Listings [0002] This application includes a Sequence Listing, which was generated on December 18, 2017, named 2873.273PC01_SequenceListing_ST25.text, and is 215,523 bytes in size. This Sequence Listing is incorporated by reference. technical field [0003] The present disclosure relates generally to the field of recombinant antibody production. More particularly, the present disclosure relates to recombinant antibodies that specifically bind human IL-15 (uncomplexed or complexed with IL-15 receptor alpha). Background technique [0004] Various references are cited throughout the specification, including patents, published patent applications, technical articles, sequence accession numbers, and other references. Each such reference is incorporated by reference in its entirety for all purposes. [0005] The cytokine interleukin 15 (IL-15), a member of the IL-2 superfamily, is produced by many cell types and tissues including monocytes, macrop...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/24G01N33/53A61P19/02
CPCG01N33/6869G01N2333/5443A61P19/02C07K16/244A61K2039/505C07K2317/21C07K2317/55C07K2317/567C07K2317/76C07K2317/92A61P1/00A61P37/04A61P17/06A61P3/10A61P37/00C07K2317/565A61P29/02A61P3/00
Inventor 大卫·约瑟·西蒙·莱恩马修·波拉德安东尼·杰拉德·道尔林恩·多萝西·普尔顿亚当·威廉·克拉克
Owner 赛福伦有限责任公司
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