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Fibronectin type III domain-based fusion proteins

a fibronectin type and domain technology, applied in the field of antibody mimetics, can solve the problem of critical delivery problem

Active Publication Date: 2019-08-20
DUKE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, delivery is a critical issue for effectively translating a protein therapeutic to the clinic.

Method used

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  • Fibronectin type III domain-based fusion proteins
  • Fibronectin type III domain-based fusion proteins
  • Fibronectin type III domain-based fusion proteins

Examples

Experimental program
Comparison scheme
Effect test

example 1

Design of Multivalent Protein-ELP Fusions

[0103]The fusion proteins included two parts (FIG. 1): (i) a multivalent targeting component (e.g., TRAILR-2 agonist or EGFR antagonist) protein in which one or more scaffold protein units (e.g., SEQ ID NO: 1 and 2 or 5) are linked by glycine-serine flexible (e.g., SEQ ID NO: 3) or structured proline-containing linkers (e.g., SEQ ID NO: 4); and (ii) an elastin-like-polypeptide connected to the multivalent protein (e.g., SEQ ID NO: 7-9).

[0104]The fusion of (i) to (ii) was at the N- or C-terminus or (ii) was interspersed among (i).

example 2

Design and Preparation of Multivalent Protein-ELP Expression Constructs

[0105]The DNA encoding the TRAILR-2-specific Tn3 unit (SEQ ID NO: 13; Swers et al., Mol. Cancer Ther., 2013, 12, 1235-1244) and the EGFR-specific domain (SEQ ID NO: 14; Friedman, et al., J. Mol. Biol. 2008, 376, 1388-1402) were purchased as double-stranded DNA “G-blocks” from Integrated DNA Technologies (Coralville, Iowa). The Tn3 G-block (SEQ ID NO: 13) was amplified using primers “Tn3For” and “Tn3Rev” primers (SEQ ID NO: 15 and 16, respectively). The gene was purchased with a (Gly4Ser)3 linker (SEQ ID NO: 3) at the C-terminus and designed with restriction sites compatible with recursive directional (RDL) ligation for seamless cloning of oligomeric genes. The EGFR-binding G-block (SEQ ID NO: 5) was purchased such that it could be inserted into the vector (SEQ ID NO: 12) using Gibson Assembly. The G-block contained 40-50 nucleic acid bases identical to those in the vector.

[0106]Enzymes used were from New England ...

example 3

Expression and Purification of Multivalent TRAILR-2 Agonist-ELP Fusion Proteins

[0108]The multivalent ELP-(Tn3)6 fusion constructs (SEQ ID NO: 10 and 11; FIG. 2) were transformed into BL21(DE3) cells (EMD / Novagen, Gibbstown, N.J.) for expression. Transformants were grown in Terrific Broth (TB) containing 45 μg / mL kanamycin and incubated overnight at 37° C. with shaking. Overnight cultures were diluted 1 to 40 into TB containing 45 μg / mL kanamycin and incubated at 37° C. with shaking for 5-8 hours. Protein expression was then induced by addition of IPTG to 1 mM, and incubation was resumed at 37° C. with shaking. In a specific embodiment, the Tn3-ELP fusion proteins were purified from the cell lysate using inverse transition cycling (ITC) as previously described (Christensen et al., Protein Science 2009, 18, 1377-1387; Hassouneh et al., Methods Enzymol. 2012, 502, 215-37). In another embodiments, C-terminally His8-tagged ELP-Tn3 fusion proteins were purified from the periplasmic extrac...

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Abstract

Provided herein are fusion proteins including at least one binding polypeptide and at least one unstructured polypeptide. The fusion protein may further include at least one linker. Further provided are methods for determining the presence of a target in a sample, methods of treating a disease, methods of diagnosing a disease in a subject, and methods of determining the effectiveness of a treatment for a disease in a subject. The methods may include administering to the subject an effective amount of the fusion protein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present patent application is a national stage filing under 35 U.S.C. § 371 of International Application No. PCT / US2016 / 024202, filed on Mar. 25, 2016, which claims the benefit of United States Provisional Application No. 62 / 138,847, filed Mar. 26, 2015, the content of which are incorporated herein by reference in their entirety.SEQUENCE LISTING[0002]The sequence listing is filed with the application in electronic format only and is incorporated by reference herein. The sequence listing text file “028193-9193-US01_As_Filed_Sequence_Listing.txt”, was created on Aug. 15, 2017, and is 46,836 bytes in size.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH[0003]This invention was made with government support under grant RO1 EB007205, 2032358, and 2032363 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD[0004]The disclosure relates to antibody mimetics and, more particularly, to fusions of...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A61K38/16C07K14/62A61K38/39C07K14/78A61K9/00A61P35/00A61K38/00
CPCC07K14/78A61K9/0019A61P35/00C07K2319/74C07K2319/00C07K2319/01A61K38/00
Inventor CHILKOTI, ASHUTOSHMANZARI, MANDANAFEVRE, MAREVA
Owner DUKE UNIV
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