Single-chain cd27-receptor agonist proteins

a single-chain, receptor-like technology, applied in the direction of peptide/protein ingredients, immunological disorders, metabolism disorders, etc., can solve the problems of difficult preparation of trimeric complexes of tnf superfamily cytokines from recombinant monomeric units, large molecular weight of trimerization domains, and inefficient trimerization, etc., to achieve short in vivo half-life and low proteolytic degradation

Inactive Publication Date: 2021-03-04
APOGENIX AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]The present invention provides specific CD27 receptor agonist proteins that mimic the CD27:CD27L interaction in vivo, exhibit low proteolytic degradation and a shorter in vivo half-life as compared to agonistic monoclonal antibodies.

Problems solved by technology

Trimeric complexes of TNF superfamily cytokines, however, are difficult to prepare from recombinant monomeric units.
A disadvantage of these fusion proteins is, however, that the trimerization domain usually has a large molecular weight and / or that the trimerization is rather inefficient.
It was found, however, that the orientation of the polypeptide chains to each other, e.g. parallel or antiparallel orientation, can hardly be predicted.
Further, the optimal number of heptad-repeats in the coiled-coil zipper motif are difficult to determine.
In addition, coiled-coil structures have the tendency to form macromolecular aggregates after alteration of pH and / or ionic strength.
Recent experiments, however, have shown that these single-chain fusion polypeptides show undesired aggregation.

Method used

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  • Single-chain cd27-receptor agonist proteins
  • Single-chain cd27-receptor agonist proteins
  • Single-chain cd27-receptor agonist proteins

Examples

Experimental program
Comparison scheme
Effect test

example 1

Manufacture of a CD27 Receptor Agonist Protein

[0141]1.1 Polypeptide Structure

[0142]A) Amino acids Met1-Gly20[0143]Ig-Kappa-signal peptide, assumed signal peptidase cleavage site after amino acid Gly 20.

[0144]B) Amino acids Glu21-Pro163[0145]First soluble cytokine domain of the human CD27L ligand (CD27L, amino acid 51-193 of SEQ ID NO: 1).

[0146]C) Amino acids GIy164-Ser 171[0147]First peptide linker element of SEQ ID NO: 2.

[0148]D) Amino acids Glu172-Pro314[0149]Second soluble cytokine domain of the human CD27L ligand (CD27L, amino acids 51-193 of SEQ ID NO: 1).

[0150]E) Amino acids GIy315-Ser322.[0151]Second peptide linker element of SEQ ID NO: 2.

[0152]F) Amino acids Glu323-Pro465[0153]Third soluble cytokine domain of the human CD27L ligand (CD27L, amino acids 51-193 of SEQ ID NO: 1).

[0154]G) Amino acids Gly466-Cys486[0155]Hinge-linker element of SEQ ID NO: 16.

[0156]H) Amino acids Pro487-Lys704[0157]Antibody Fc fragment domain of SEQ ID NO: 13.

[0158]The above CD27 receptor agonist pr...

example 2

Expression and Purification

[0165]2.1 Cloning, Expression and Purification of Fusion Polypeptides

[0166]The aforementioned fusion proteins are expressed recombinantly in two different eukaryotic host cells employing the methods described below:

[0167]Method for Small Scale Expression of of CD27 Receptor Agonist Fusion Proteins:

[0168]For initial analysis of aforementioned CD27 receptor agonist fusion proteins, Hek293 cells grown in DMEM+GlutaMAX (GibCo) supplemented with 10% FBS, 100 units / ml Penicillin and 100 [mu]g / ml Streptomycin are transiently transfected with a plasmid containing an expression cassette for a fusion polypeptide and an appropriate selection marker, e.g. a functional expression cassette comprising a blasticidine, puromycin or hygromycin resistence gene. In those cases, where a plurality of polypeptide chains is necessary to achieve the final product, the expression cassettes will be either combined on one plasmid or positioned on different plasm ids during the transf...

example 3

SDS-PAGE Results of Dimer Proteins Expressed from Protein A

[0179]To determine if the homodimer of Protein A is covalently linked, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) experiments were performed under reducing and non-reducing conditions. The size of the main band under reducing conditions is only about half of the size as observed under non-reducing conditions. This indicates that the homodimer is covalently linked by disulfide bridges (see also FIG. 7).

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Abstract

Provided herein are specific CD27 receptor agonist proteins, nucleic acids encoding the same, and methods of treating a subject having a CD27L-associated disease or disorder. The CD27 receptor agonist proteins provided herein comprise three soluble CD27L domains an and Fc fragment. The CD27 receptor agonist proteins are substantially non-aggregating and suitable for therapeutic, diagnostic and / or research applications.

Description

[0001]This application is a divisional of U.S. application Ser. No. 15 / 956,964, filed Apr. 19, 2018; which is a continuation of PCT / EP2016 / 075579, filed Oct. 24, 2016; which claims priority to U.S. Provisional Application No. 62 / 245,689, filed Oct. 23, 2015. The contents of the above applications are incorporated herein by reference in their entirety.REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM[0002]The Sequence Listing is concurrently submitted herewith with the specification as an ASCII formatted text file via EFS-Web with a file name of Sequence_Listing.txt with a creation date of Apr. 13, 2018, and a size of 128 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.FIELD OF THE INVENTION[0003]The present invention provides specific CD27 receptor agonist proteins comprising three soluble CD27L domains and an Fc fragment, nucleic acid molecules encoding the CD27 receptor agonist pro...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/705C07K16/28
CPCC07K14/70575C07K16/2818C07K16/2809A61K38/00C07K2319/30C07K2319/32C07K2319/22C07K2319/00C07K2319/35C07K2319/74A61P19/02A61P25/00A61P29/00A61P3/00A61P31/00A61P35/00A61P35/02A61P37/06
Inventor GIEFFERS, CHRISTIANHILL, OLIVERTHIEMANN, MEINOLFSCHNYDER, TIM
Owner APOGENIX AG
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