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Masked cytokine polypeptides

A technology of cytokines and masking parts, applied in the field of masked cytokine peptides, can solve the problems of leakage, adverse health outcomes, etc.

Pending Publication Date: 2021-08-20
西里欧发展公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, administration of high doses of cytokines may induce adverse health outcomes such as vascular leakage through systemic immune activation

Method used

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  • Masked cytokine polypeptides
  • Masked cytokine polypeptides
  • Masked cytokine polypeptides

Examples

Experimental program
Comparison scheme
Effect test

example 1

[2775] Example 1: Engineering of Masked IL-2 Polypeptides and Masked IL-15 Polypeptides

[2776] Masked IL-2 polypeptide constructs and masked IL-15 polypeptide constructs were generated according to the teachings herein. In the examples that follow, some experiments involved the use of masked IL-2 and IL-15 polypeptide constructs in monomeric form and some experiments involved the use of masked IL-2 and IL-15 polypeptide constructs in dimeric form , such as a dimer formed by disulfide-bonding two copies of the same masked polypeptide construct (homodimer), or a heterodimer formed by two different polypeptides (see, e.g., Table 8 -11).

[2777] Generate masked IL-2 polypeptide constructs comprising IL-2 polypeptides or functional fragments thereof, masking moieties and half-life extending domains, such as albumin, antibodies or fragments thereof (e.g., Fc region, heavy chain and / or light chain ), an albumin binding peptide, an IgG binding peptide or a polyamino acid sequen...

example 2

[2817] Example 2: In vitro characterization of masked IL-2 and IL-15 polypeptides

[2818] Several in vitro cellular and functional assays were employed to characterize the masked IL-2 polypeptide constructs and masked IL-15 polypeptide constructs generated in Example 1.

[2819] produce

[2820] Plasmids encoding the constructs (eg, masked IL-2 polypeptide construct and masked IL-15 polypeptide construct) were transfected into Expi293 cells (Life Technologies A14527) or HEK293-6E cells (National Research Council; NRC). 1 mg of total DNA was used for transfection at a 1:1 ratio of PEIpro (Polyplus Transfection, 115-100) to total DNA. DNA and PEI were each added to 50 mL of OptiMem (Life Technologies 31985088) medium and sterile filtered. DNA and PEI were pooled for 10 minutes and added to Expi293 cells or HEK293 cells respectively, for expi293 cells at a cell density of 1.8-2.8x10 6 cells / mL or 0.85-1.20x10 6 cells / m with a viability of at least 95%. Following the same ...

example 3

[2865] Example 3: In vivo characterization of masked IL-2 and IL-15 polypeptides

[2866] Pharmacokinetics

[2867] The pharmacokinetics of the masked IL-2 polypeptide constructs and masked IL-15 polypeptide constructs generated in Example 1 were evaluated in vivo using a mouse model.

[2868] Mice are treated intravenously or subcutaneously with the construct, and plasma concentrations of the construct are measured over time. In some experiments, some mice were treated with a control for comparison. In some experiments, some mice were treated with aldesleukin as a control for masking IL-2 polypeptide treatment. In some experiments, the treated mice had tumors. In some experiments, the treated mice were tumor-free. In some experiments, mice are treated with the construct and blood is drawn at various times during treatment, which may include drawing blood prior to initiation of treatment and processing it to obtain plasma. In some experiments, blood was drawn at various...

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Abstract

Provided herein are cytokines or functional fragments thereof that, in some embodiments, are engineered to be masked by a masking moiety at one or more receptor binding site(s) of the cytokine or functional fragment thereof. In some embodiments, the cytokines are engineered to be activatable by a protease at a target site, such as in a tumor microenvironment, by including a proteolytically cleavable linker. In some embodiments, the proteolytically cleavable linker links the cytokine to the masking moiety, links the cytokine to a half-life extension domain, and / or links the masking moiety to a half-life extension domain. The masking moiety blocks, occludes, inhibits (e.g., decreases) or otherwise prevents (e.g., masks) the activity or binding of the cytokine to its cognate receptor or protein. Upon proteolytic cleavage of the cleavable linker at the target site, the cytokine becomes activated, which renders it capable of binding to its cognate receptor or protein with increased affinity.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application No. 62 / 737,803, filed September 27, 2018, U.S. Provisional Application No. 62 / 888,276, filed August 16, 2019, and U.S. Provisional Application No. 62 / 891,199, filed August 23, 2019 priority. The contents of the applications listed above are hereby incorporated by reference in their entirety for all purposes. [0003] Incorporated by reference into the sequence listing [0004] This application is filed together with a Sequence Listing in electronic format. The sequence listing is provided as a file named 737762000940SeqList created on September 26, 2019 and has a size of 1,024.111 KB. The information in the sequence listing in electronic format is incorporated by reference in its entirety. technical field [0005] The present invention relates to masked cytokines and methods relating to their use and preparation. Background technique [0006] Cancer is the secon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/52C07K14/54
CPCC07K14/52C07K14/54C07K14/55C07K2319/50C07K2319/30C07K2319/01C07K14/5443A61K38/00A61P35/00C07K2317/52C07K2317/94A61K38/2013A61K38/2086
Inventor M·卡罗D·M·拉伊D·托马尔P·约翰逊R·罗森菲尔德R·奥哈根邱华伟
Owner 西里欧发展公司