Immunomodulatory proteins with tunable affinities
A technology of immunomodulatory proteins and immunoglobulins, which can be used in peptide/protein components, animal/human proteins, medical preparations containing active ingredients, etc., and can solve problems such as lack of important attributes
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Embodiment approach
[0279] The implementation methods provided include:
[0280] Embodiment 1. In some embodiments, an immunomodulatory protein is provided, which contains at least one non-immunoglobulin affinity modified immunoglobulin superfamily (IgSF) domain, the immunoglobulin superfamily domain containing a wild-type IgSF domain One or more amino acid substitutions in wherein: at least one affinity-modified IgSF domain has enhanced binding to at least two associated binding partners compared to the wild-type IgSF domain; and at least one affinity-modified IgSF domain is specific To non-competitively bind at least two related binding partners.
[0281] Embodiment 2. In some other embodiments of embodiment 1, at least two cognate binding partners are cell surface molecules expressed on the surface of mammalian cells.
[0282] Embodiment 3. In some other embodiments of Embodiment 2, the cell surface molecule is expressed in a cis configuration or a trans configuration.
[0283] Embodiment 4. In s...
Embodiment 1
[0477] Generation of mutant DNA constructs of IgSF domain
[0478] Example 1 describes the generation of mutant DNA constructs of human CD80, CD86, ICOSL and NKp30 IgSF domains for translation and expression on the surface of yeast as a yeast display library.
[0479] A. Degenerate library
[0480] For libraries that are fully or partially randomized with degenerate codons for specific residues of the target protein, human CD80 (SEQ ID NO: 28), ICOSL (SEQ ID NO: 32) and NKp30 (SEQ ID NO: 54) cells A collection of overlapping oligonucleotides with a length of more than 80 base pairs (bp) from the coding DNA of the outer domain (ECD) was purchased from Integrated DNA Technologies (Clover, Iowa, USA). In order to generate a library of multiple variants of each ECD, the oligonucleotide contains the required degenerate codons at the required amino acid positions. Degenerate codons are generated using the algorithm located at URL: rosettadesign.med.unc.edu / SwiftLib / .
[0481] Generally, t...
Embodiment 2
[0488] DNA library introduced into yeast
[0489] Example 2 describes the introduction of CD80, CD86, ICOSL, and NKp30 DNA libraries into yeast.
[0490] In order to introduce degenerate and random library DNA into yeast, electroporation competent cells of yeast strain BJ5464 (ATCC.org; ATCC No. 208288) were prepared, and they were basically as described on Gene Pulser II (BioRad, USA). The electroporation is performed using the electroporation ready-to-use DNA from the above steps (Colby, DW, etc., 2004 Methods Enzymology 388, 348-358). The only exception is that the transformed cells were grown in non-inducible minimal selective SCD-Leu medium to accommodate the LEU2 selectable marker carried by the modified plasmid pBYDS03.
[0491] The library size was determined by plating a dilution of freshly recovered cells on SCD-Leu agar plates, and then inferring the library size from the number of individual colonies from the plating that produced at least 50 colonies per plate. The rem...
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