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Modified membrane type serine protease 1 (mtsp-1) polypeptides and methods of use

A technology of MTSP-1 and serine protease, which is applied in the field of modified membrane-type serine protease 1 (MTSP-1) polypeptide and its application, and can solve problems such as short half-life

Pending Publication Date: 2020-05-05
VERTEX PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Small molecules have short half-lives in the body and require constant infusion to maintain complement inhibition, limiting their effects, especially in chronic diseases

Method used

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  • Modified membrane type serine protease 1 (mtsp-1) polypeptides and methods of use
  • Modified membrane type serine protease 1 (mtsp-1) polypeptides and methods of use
  • Modified membrane type serine protease 1 (mtsp-1) polypeptides and methods of use

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Experimental program
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preparation example Construction

[0660] The preparation of fusions of therapeutic proteins with other moieties such as PEGylation, conjugation to albumin, targeting moieties such as antibodies and antigen-binding fragments thereof, immunoglobulins, fc fusions, and Fusions of albumin (HSA), XTEN fusion proteins, modification of glycosylation patterns (for a review of various fusion proteins used to improve the pharmacokinetic properties of therapeutic proteins, see Strohl (2015) BioDrugs 29:215 -239). Any of these known modes for improving the pharmacological properties of therapeutic agents can be applied to the modified MTSP-1 polypeptides provided herein.

[0661] Fusion proteins comprising a modified MTSP-1 polypeptide provided herein and one or more other polypeptides are also provided. Pharmaceutical compositions containing such fusion proteins formulated for administration by appropriate routes are provided. Fusion proteins are formed by linking a modified MTSP-1 polypeptide and another polypeptide (s...

Embodiment 1

[0769] Cloning and Expression of Modified MTSP-1 Polypeptides, and Screening for Modified MTSP-1 Polypeptides Cleaving C3 at Target Sites

[0770] A. Cloning and mutagenesis of MTSP-1

[0771] A nucleic acid encoding amino acids 615-855 having the C122S substitution of the human MTSP-1 polypeptide shown in SEQ ID NO: 1 was prepared. The construct includes a proregion, an activation sequence, and a protease domain, and contains residues 598 to C of the sequence disclosed by Takeuchi et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 11054 and SEQ ID NO: 1 terminal (ie, corresponding to residues 598 to 855 of the amino acid sequence shown in SEQ ID NO: 1).

[0772] Modified MTSP-1 polypeptides were generated by Quikchange site-directed mutagenesis (Stratagene) according to the manufacturer's instructions with specially designed oligonucleotides that served as primers to introduce designed mutations into newly synthesized inside the DNA. Briefly, PCR sample reactions were set up ...

Embodiment 2

[0820] In vitro cleavage of complement protein C3

[0821] The activity of the modified MTSP-1 polypeptide was determined by cleavage of the substrate complement protein human C3 by measuring the amount of intact human C3 remaining after incubation with various concentrations of protease for 1 hour at 37°C. In this assay, signal is generated in the presence of intact human C3 and lost upon C3 cleavage.

[0822] 2 μM plasma-purified human C3 (hC3; Complement Technologies; Tyler, TX) was incubated with modified MTSP-1 polypeptides (0–250 nM) at 37° C. in the presence of 50 mM Tris, pH 8.0, 50 mM NaCl and 0.01% Tween -20 buffer for 1 hour. The activity of the modified MTSP-1 polypeptide was quenched by adding EGR-CMK (Haematologic Technologies, EGRCK-01) to a final concentration of 10 μM, and the hC3 / modified MTSP-1 polypeptide mixture was allowed to stand at ambient temperature for 30 minute.

[0823] Using Amplified Luminescent Proximity Homogeneous Assay Screen ( Perkin El...

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Abstract

Provided are MTSP-1 polypeptides modified to have altered activity and / or specificity so that they cleave a complement protein, such as complement protein C3, to inhibit its activity and thereby inhibit complement activation. The modified MTSP-1 polypeptides that inhibit complement activation can be used for treatment of diseases and conditions in which complement activation plays a role. Such diseases and conditions include inflammatory diseases and diseases with an inflammatory component. Exemplary of these disorders are ischemic and reperfusion disorders, including myocardial infarction andstroke, sepsis, autoimmune diseases, ophthalmic disorders, such as diabetic retinopathies and macular degeneration, including age-related macular degeneration (AMD), and transplanted organ rejection,such as renal delayed graft function (DGF).

Description

[0001] related application [0002] Claiming the benefit of priority to U.S. Provisional Application Serial No. 62 / 523,735, filed June 22, 2017, to Madison, Edwin L., Soros, Vanessa, and Popkov, Mikhail, entitled "MODIFIED MEMBRANE TYPE SERINE PROTEASE 1 (MT-SP1) POLYPEPTIDES AND METHODS OFUSE". The benefit of priority of U.S. Provisional Application Serial No. 62 / 664,051 filed April 27, 2018 to Madison, Edwin L., Soros, Vanessa, and Popkov, Mikhail, entitled "MODIFIED MEMBRANE TYPE SERINE PROTEASE 1 (MTSP-1) POLYPEPTIDES AND METHODS OFUSE". Where permitted, the respective subject matter of these applications are incorporated by reference in their entirety. [0003] Electronically provided sequence listing incorporated by reference [0004] An electronic version of the Sequence Listing is submitted along with it, the contents of which are incorporated by reference in their entirety. The electronic file was created on June 18, 2018, is 1.4 megabytes in size, and is titled ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48C07K14/435
CPCC07K14/435A61K38/00A61K45/06C12Y304/21109A61K31/397C12N9/6424A61K2300/00
Inventor E·L·麦迪逊V·绍罗什M·波普科夫
Owner VERTEX PHARMA INC
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