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Therapeutic interferon alpha 1 proteins

A technology of interferon alpha, chimeric protein, applied in interferon, immunoglobulin, cytokine/lymphokine/interferon, etc., can solve the problem of lack of general interest and pursuit

Pending Publication Date: 2021-12-07
ORIONIS BIOSCI INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These features, among others, have contributed to the long-standing lack of general interest and pursuit of IFNα1 as a potential therapeutic agent
The efficacy of type I interferons in clinical practice is limited by ineffective dosing due to significant systemic toxicity and side effects, including influenza-like syndrome, depression, hepatotoxicity, autoimmune disease, thyroid dysfunction, and weight loss

Method used

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  • Therapeutic interferon alpha 1 proteins
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  • Therapeutic interferon alpha 1 proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0670] Example 1: Generation, Production and Purification of IFNα1 AcTaferon (AFN)

[0671] To generate AFN based on IFNα1 fusion protein, the nucleic acid sequence encoding IFNα1 was ligated via the nucleic acid sequence encoding the flexible 20*GGS flexible linker to the nucleic acid encoding the VHH targeting human CD20 in the pHEN6C vector (under the control of the PelB signal peptide) Sequences were used for bacterial expression. Finally add His 6 tag for purification.

[0672] AFN expression was induced overnight with 1 mM IPTG, cells were pelleted, and periplasmic extracts were prepared using TES (0.2M Tris pH 8.0, 0.5mM EDTA, 0.5M sucrose) and TES / 4 buffer. Proteins were purified from extracts using TALON Metal affinity resin according to the manufacturer's guidelines, and imidazole was removed from samples using a PD10 column (GEHEALTHCARE).

[0673] A similar process was used to generate IFN-α2-based AFN.

[0674] Structure and sequence of IFNα1 AFN

[0675]...

Embodiment 2

[0685] Example 2: Method of STAT1 Phosphorylation in Peripheral Blood Mononuclear Cells (PBMC) and IFN Response in HL116 Cells A reporter gene activity

[0686] PBMCs were isolated from buffy coats of healthy donors by density gradient centrifugation using Lymphoprep (STEMCELL TECHNOLOGIES). Cells were washed twice with FACS buffer (2% FBS, 1 mM EDTA in PBS) and stained with anti-human CD20 FITC (SINOBIOLOGICALS) for 20 min at 4°C. After washing twice, cells were stimulated with serial dilutions of wild-type IFNα2, CD20 VHH-IFNα2, IFNα1 and CD20 VHH-IFNα1 for 15 minutes at 37°C. After fixation (10 min, 37°C, Fixation Buffer I; BD BIOSCIENCES), permeabilization (30 min, on ice, Perm III Buffer I; BDBIOSCIENCES) and washing, cells were treated with anti-STAT1 pY701 Ab (BD Biosciences )dyeing. Samples were collected using a Macsquant X instrument (MILTENYI BIOTEC) and analyzed using FlowLogic software (MILTENYI BIOTEC). Induction of pSTAT1 mirrors activation of IFNAR by in...

Embodiment 3

[0687] Example 3: STAT1 Phosphorylation in Peripheral Blood Mononuclear Cells (PBMC) and IFN Response Reporting in HL116 Cells gene activity

[0688] Figure 21A-Figure 21D , Figure 22A-Figure 22D and the data in Table 7 clearly show that incorporation of wild-type IFNα1 into a chimeric fusion protein (exemplified here by linking IFNα1 to an anti-CD20 VHH) results in reduced IFNAR stimulating activity of IFNα1 compared to wild-type IFNα1 (both PBMC and HL116 as shown here for CD20 negative cells). However, IFNα1 activity was induced / restored specifically on target cells (CD20 positive, both PBMC and HL116-huCD20). Importantly, activation of IFNAR signaling is highly selective for targeted (CD20-positive) cells versus non-targeted (CD20-negative) cells, with approximately 200- to 600-fold targeting selectivity. In contrast, in the case of IFNα2, the targeting selectivity observed in CD20-positive versus CD20-negative PBMCs and HL116 cells was only 20–60-fold, thus compa...

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Abstract

The present invention relates, in part, to chimeric proteins or chimeric protein complexes, such as Fc-based chimeric protein complexes, comprising interferon alpha 1, or a variant thereof, and their use as therapeutic agents.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 906,431, filed September 26, 2019, and U.S. Provisional Patent Application No. 62 / 825,569, filed March 28, 2019, the contents of which are hereby incorporated by reference The method is integrated as a whole. technical field [0003] The present invention relates, in part, to chimeric proteins or chimeric protein complexes (including Fc-based chimeric protein complexes) comprising interferon alpha 1 (IFNα1 ) or variants thereof and their use as therapeutic agents. [0004] sequence listing [0005] The contents of the text file electronically filed with this document are hereby incorporated by reference in their entirety. A computer-readable copy of the sequence listing (file name: ORN-057PC_Sequence_Listing_ST25, date of record: March 26, 2020; file size: 385,024 bytes). Background technique [0006] Type I interferons (I...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/00A61P35/00A61P35/02A61P31/00A61P37/02A61P9/00A61P17/02A61P9/10A61P25/00A61P3/00
CPCA61K38/00C07K14/56C07K2319/70C07K16/2887C07K2317/569C07K2319/00C07K2319/30C07K2317/92C07K16/2818C07K16/2827C07K16/2851C07K2317/52C07K2317/22C07K2317/31C07K2317/24
Inventor N·克雷E·德普拉L·扎比奥J·塔威尼尔
Owner ORIONIS BIOSCI INC
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