Ctla-4 variant immunomodulatory proteins and uses thereof

A technology of CTLA-4, variants, applied in the field of preparation and use of compositions of such proteins

Pending Publication Date: 2020-10-20
ALPINE IMMUNE SCI INC
View PDF91 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although therapeutics that can modulate IS are

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Ctla-4 variant immunomodulatory proteins and uses thereof
  • Ctla-4 variant immunomodulatory proteins and uses thereof
  • Ctla-4 variant immunomodulatory proteins and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0679] Generation of mutant DNA constructs of the IgSF domain

[0680] A mutant DNA construct of the human CTLA-4 IgSF domain was generated for translation and expression on the surface of yeast as a yeast display library.

[0681]A library containing random amino acid substitutions was constructed to identify variants of the ECD of CTLA-4 based on the wild-type human CTLA-4 sequence shown in SEQ ID NO: 2 below: .

[0682] KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGINGTQIYVIDPEPPCPDSD (SEQ ID NO: 2)

[0683] DNA encoding wild-type CTLA-4 ECD was cloned between the BamHI and KpnI sites of the modified yeast display vector pBYDS03 (Life Technologies). By error-prone PCR, using supplemented with MnCl 2 Genemorph II Kit (Agilent, USA) and ECD-specific oligonucleotides that overlapped the pBYDS03 cloning vector by 40 bp outside and including the BamHI cloning site and the KpnI cloning site to introduce mutations. Th...

example 2

[0686] Introduction of DNA library into yeast

[0687] The CTLA-4 DNA library generated in Example 1 was introduced into yeast using electroporation. Briefly, electroporation-competent cells of yeast strain BJ5464 (ATCC.org; ATCC No. 208288) were prepared and tested on a Gene Pulser II (Biorad) Electroporation was performed on DNA prepared for electroporation as described above (Colby, D.W. et al., 2004 "Methods Enzymology" 388, 348-358). The only exception is that transformed cells were grown in non-inducible minimally selective SCD-Leu medium to accommodate the LEU2 selectable marker carried by the modified plasmid pBYDS03. One liter of SCD-Leu medium consists of 14.7 g sodium citrate, 4.29 g citric acid monohydrate, 20 g dextrose, 6.7 g yeast nitrogen base and 1.6 g yeast synthesis deficient medium supplement without leucine (yeast synthetic drop-out media supplement). Before use, filter-sterilize the medium using a 0.22 μm vacuum filter unit.

[0688] The library size ...

example 3

[0690] yeast selection

[0691] Yeast expressing affinity-modified variants of CTLA-4 are selected for ICOSL and / or CD86.

[0692] A number of cells equal to at least 10 times the estimated library size were thawed from individual library stocks, suspended to 1.0 x 10E6 cells / ml in non-inducing SCD-Leu medium, and grown overnight. The next day, an amount of cells equal to 10 times the library size was centrifuged at 2000 RPM for 2 minutes and resuspended to 5.0 x 10E6 cells / ml in inducible SCDG-Leu medium. One liter of SCDG-Leu induction medium consists of 5.4 g of Na dissolved in water 2 HPO 4 , 8.56 g NaH 2 PO 4 ·H 2 0. 20 g galactose, 2.0 g dextrose, 6.7 g yeast nitrogen base and 1.6 g yeast synthesis deficient medium supplement without leucine and sterilized through a 0.22 μm membrane filter unit. Cultures were grown in induction medium at room temperature for 1 day to induce expression of library proteins on the surface of yeast cells.

[0693] The induced yeast li...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

Provided herein are variant CTLA-4 polypeptides and immunomodulatory proteins and nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of disease applications, including for treatment of autoimmune or inflammatory conditions. Compositions and methods for making and using such proteins are provided.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application No. 62 / 733,615, filed September 19, 2018, U.S. Provisional Application No. 62 / 613,379, filed January 3, 2018, and U.S. Provisional Application No. 62 / 613,379, filed October 10, 2017. The content of each of said US Provisional Applications, the priority benefit of Application Serial No. 62 / 570,619, is hereby incorporated by reference in its entirety. [0003] Incorporated by reference into the sequence listing [0004] This application is filed with a sequence listing in electronic format. The sequence listing is provided in a file named 761612002040SeqList.txt created on October 8, 2018, with a size of 1,138,300 bytes. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. technical field [0005] The present disclosure relates to immunomodulatory proteins including variant CTLA-4 and nucleic acids encoding ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C07K14/705A61K38/16A61K38/17
CPCA61K38/00C07K14/70521C07K2319/30A61P35/00A61P37/02A61K47/65A61P19/02A61K35/17C07K14/7051C07K2319/02C07K2319/03C12N5/0637C12N15/00
Inventor L·埃文斯J·L·库伊普R·斯汪森
Owner ALPINE IMMUNE SCI INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap