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Fusion toxin proteins for treating diseases associated with CMV infection

A technology of toxins and immunotoxins, applied in the direction of targeting specific cell fusions, viruses, antiviral agents, etc.

Pending Publication Date: 2022-03-18
UNIVERSITY OF COPENHAGEN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A challenge in designing potent immunotoxins targeting the US28 receptor is the existence of a human cognate receptor named CX3CR1

Method used

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  • Fusion toxin proteins for treating diseases associated with CMV infection
  • Fusion toxin proteins for treating diseases associated with CMV infection
  • Fusion toxin proteins for treating diseases associated with CMV infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0257] Example 1 - Preparation of fusion proteins SYN000, SYN001, SYN003 and SYN004

[0258] structure of immunotoxin

[0259] Disclosed herein is a series of immunotoxins designed to specifically target US28. They are based on the chemokine CX3CL1 (fractalkine) and exotoxin A from Pseudomonas aeruginosa (both as Figure 7 shown). Drug candidates SYN000 (SEQ ID NO: 17), SYN001 (SEQ ID NO: 18), SYN003 (SEQ ID NO: 19) and SYN004 (SEQ ID NO: 10) are all single-unit structures consisting of approximately 400 amino acids and domains polypeptide chains, such as Figure 8 shown.

[0260] Production of immunotoxins

[0261] Immunotoxins are produced in E. coli as insoluble protein aggregates (inclusion bodies, IB). The drug substance manufacturing process consists of three processing stages: cell culture and harvesting, recovery and purification. The E. coli culture step is to produce IBs containing high levels of drug substances. IB was recovered by a series of washes and c...

Embodiment 2

[0262] Example 2 - Affinity (K i value)

[0263] Receptor competition for binding

[0264] Stable inducible clones of US28-HEK293 cells and CX3CR1-HEK293 cells in 10% CO 2 and 37°C in a humidified incubator, grown in Dulbecco's modified Eagle's medium (DMEM) GlutaMAX (GIBCOR) containing 10% fetal bovine serum and 180 units / mL penicillin and 45 μg / mL streptomycin. Cells were seeded at 10,000 cells per well in 100 μL of growth medium in poly-D-lysine (Invitrogen)-coated 96-well tissue culture plates (Nunc). One day after seeding, US28 and CX3CR1 expression was induced by tetracycline (5 ng / mL) to obtain 5-10% specific binding. One day after induction, competition binding studies were performed in duplicate. Briefly, cells were washed twice in binding buffer consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (50 mM HEPES, 5 mM MgCl) 2 , 1mM CaCl 2 , pH7.2) and 0.5% bovine serum albumin. Cells were then incubated at 4°C with ~25pM 125 I-CX3CL...

Embodiment 3

[0274] Example 3 - SYN000, SYN001, SYN003 and SYN004 pair induced US28 HEK293 cells and induced Potency in CX3CR1 HEK293 cells

[0275] In vitro potency

[0276] Stable inducible clones of US28-HEK293 cells and CX3CR1-HEK293 cells in 10% CO 2and 37°C in a humidified incubator, grown in Dulbecco's modified Eagle's medium (DMEM) GlutaMAX (GIBCOR) containing 10% fetal bovine serum and 180 units / mL penicillin and 45 μg / mL streptomycin. Cells were seeded at 11,000 cells per well in 100 μL of growth medium in poly-D-lysine (Invitrogen)-coated 96-well tissue culture plates (Nunc). Twenty-four hours after inoculation, receptor expression was induced with 0.125 μg / mL (US28) and 0.5 μg / mL (CX3CR1). One day after receptor induction, various concentrations of the indicated immunotoxins (0.01 μM–0.1 pM) and buffers (mock treatment) were added to growth medium in a final volume of 100 μL and incubated at 37°C for 24 hours. To estimate cell viability, AlamarBlue (Invitrogen) was mix...

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Abstract

The present invention relates to immunotoxins for use in the treatment of diseases associated with CMV infection. The invention also relates to the use of the immunotoxin and a pharmaceutical composition comprising the immunotoxin as a medicament, as well as a kit comprising the immunotoxin for the treatment or prevention of CMV infection.

Description

technical field [0001] The present invention relates to immunotoxins for treating diseases associated with CMV infection. The present invention also relates to the use of the immunotoxin and the pharmaceutical composition comprising the immunotoxin as a medicine, and a kit for treating or preventing CMV infection comprising the immunotoxin. Background technique [0002] Cytomegalovirus [0003] Cytomegalovirus (CMV) is an important human pathogen and a major opportunist that causes disease in immunocompromised subjects such as AIDS (AIDS) patients, newborns and those who have received immunosuppressive drugs (e.g. as a transplant part of the program) individuals. In these individuals, the consequences of CMV in recurrent and / or acute infections can be extremely serious, including retinitis, encephalitis, and pneumocystis and other pathologies. Furthermore, in immunocompetent hosts, CMV establishes a persistent lifelong infection through which it is associated with various...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/16A61K45/06A61K47/64A61P31/22
CPCC07K14/21C07K14/521A61K47/642A61K45/06A61K38/164A61P31/22C07K2319/33C07K2319/55A61K38/00A61K2300/00C12N2710/16111A61P31/12
Inventor 托马斯·尼奇克·克莱达尔梅特·玛丽·罗斯基尔德
Owner UNIVERSITY OF COPENHAGEN
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