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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
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although therapeutics that can modulate IS are known, improved therapeutics are still needed

Method used

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  • 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...

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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

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Application Information

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IPC IPC(8): C07K14/705A61K38/16A61K38/17
CPCA61K38/00C07K14/70521C07K2319/30C12N5/0637A61P35/00A61P37/02A61K47/65A61P19/02A61K35/17C07K14/7051C07K2319/02C07K2319/03C12N15/00
Inventor L·埃文斯J·L·库伊普R·斯汪森
Owner ALPINE IMMUNE SCI INC
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