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JP-2026514409-A - IdeS variant protein and method of using the same

JP2026514409AJP 2026514409 AJP2026514409 AJP 2026514409AJP-2026514409-A

Abstract

This disclosure relates, as a whole, to variant IdeS polypeptides having lower immunogenicity and maintaining or improving IdeS stability and IgG cleavage activity, as well as to methods for using these variant IdeS polypeptides.

Inventors

  • ザウアー，マクシミリアン
  • シエカー，デイビッド
  • ディヴェイン，グレイス
  • タッチャ，エリック
  • ソン，イファン
  • プロクコ，エリック
  • ファレル，ダニエル
  • チャン，クイ
  • ドゥッツァー，ベンジャミン
  • フレイジャー，エミリー
  • レウワーキ，リンカーン
  • ロバートソン，ギャビン
  • アグホブ，アーロン
  • チャン，ジェリー

Assignees

  • サイラス バイオテクノロジー，インコーポレイテッド

Dates

Publication Date
20260511
Application Date
20240329
Priority Date
20230329

Claims (20)

  1. An IdeS variant protein comprising at least two modifications at positions selected from the group consisting of 68, 75, 166, 187, 213, 236, 277, 302, 303, 306, and 318 of SEQ ID NO: 1.
  2. The modification at position 68 is a substitution of valine to threonine, as described in claim 1, for the IdeS variant protein.
  3. The modification at position 75 is a substitution of alanine to proline, as described in claim 1 or 2, for the IdeS variant protein.
  4. The IdeS variant protein according to any one of claims 1 to 3, wherein the modification at position 166 is a substitution of threonine to arginine, or a substitution of threonine to glycine.
  5. The IdeS variant protein according to any one of claims 1 to 4, wherein the modification at position 187 is a substitution of serine to aspartic acid, or a substitution of serine to glutamic acid.
  6. The modification at position 213 is a substitution of threonine to glutamic acid, as described in any one of claims 1 to 5, for the IdeS variant protein.
  7. The modification at position 236 is a substitution of serine to cysteine, as described in any one of claims 1 to 6, for the IdeS variant protein.
  8. The IdeS variant protein according to any one of claims 1 to 7, wherein the modification at position 277 is a substitution of leucine to cysteine.
  9. The IdeS variant protein according to any one of claims 1 to 8, wherein the modification at position 302 is a substitution of serine to aspartic acid, a substitution of serine to lysine, or a substitution of serine to glutamic acid.
  10. The IdeS variant protein according to any one of claims 1 to 9, wherein the modification at position 303 is a substitution of alanine to aspartic acid, a substitution of alanine to asparagine, or a substitution of alanine to glutamine.
  11. The modification at position 306 is a substitution of valine to threonine, as described in any one of claims 1 to 10, for the IdeS variant protein.
  12. The IdeS variant protein according to any one of claims 1 to 11, wherein the modification at position 318 is a substitution of isoleucine to lysine, a substitution of isoleucine to aspartic acid, or a substitution of isoleucine to glycine.
  13. The IdeS variant protein according to any one of claims 1 to 12, further comprising a modification at position 308.
  14. The IdeS variant protein according to claim 13, wherein the modification at position 308 is a substitution of isoleucine to leucine.
  15. The IdeS variant protein according to any one of claims 1 to 14, comprising a substitution of valine to threonine at position 68, alanine to proline at position 75, threonine to arginine at position 166, serine to aspartic acid at position 187, threonine to glutamic acid at position 213, serine to lysine at position 302, and alanine to aspartic acid at position 303.
  16. The IdeS variant protein according to any one of claims 1 to 14, comprising a substitution of valine to threonine at position 68, a substitution of alanine to proline at position 75, a substitution of threonine to arginine at position 166, a substitution of serine to aspartic acid at position 187, a substitution of threonine to glutamic acid at position 213, a substitution of serine to glutamic acid at position 302, and a substitution of alanine to aspartic acid at position 303.
  17. The IdeS variant protein according to any one of claims 1 to 14, comprising a substitution of valine to threonine at position 68, alanine to proline at position 75, threonine to arginine at position 166, serine to aspartic acid at position 187, threonine to glutamic acid at position 213, serine to lysine at position 302, alanine to aspartic acid at position 303, and valine to threonine at position 306.
  18. The IdeS variant protein according to any one of claims 1 to 14, comprising a substitution of valine to threonine at position 68, alanine to proline at position 75, threonine to arginine at position 166, serine to aspartic acid at position 187, threonine to glutamic acid at position 213, serine to glutamic acid at position 302, valine to threonine at position 306, and isoleucine to lysine at position 318.
  19. The IdeS variant protein according to any one of claims 1 to 14, comprising the substitution of valine to threonine at position 68, alanine to proline at position 75, threonine to arginine at position 166, serine to aspartic acid at position 187, threonine to glutamic acid at position 213, serine to cysteine at position 236, leucine to cysteine at position 277, serine to glutamic acid at position 302, alanine to aspartic acid at position 303, and valine to threonine at position 306.
  20. The IdeS variant protein according to claim 14, wherein the variant protein comprises a substitution of valine to threonine at position 68, a substitution of alanine to proline at position 75, a substitution of threonine to arginine at position 166, a substitution of serine to aspartic acid at position 187, a substitution of threonine to glutamic acid at position 213, a substitution of serine to cysteine at position 236, a substitution of leucine to cysteine at position 277, and a substitution of isoleucine to leucine at position 308.

Description

cross reference [0001] This application claims benefit and priority to U.S. Provisional Patent Application No. 63/492,891 filed on 29 March 2023 and U.S. Provisional Patent Application No. 63/584,748 filed on 22 September 2023, both of which are incorporated herein by reference in their entirety. Areas of this disclosure [0002] This disclosure relates to variant Ides polypeptides having lower immunogenicity and maintaining or improving IdeS stability and IgG cleavage activity, as well as methods for using these variant Ides polypeptides. background [0003] IdeS (an immunoglobulin-degrading enzyme derived from Streptococcus pyogenes) is a cysteine protease that catalyzes the rapid cleavage of all human IgG subclasses within the hinge region, cleaving the antigen-binding domain of IgG from the Fc region which mediates immune effector function. Separation of the antigen-binding Fab region from the Fc portion prevents Fc-mediated recruitment of immune effector functions to antigens, such as the recruitment of lethal immune cells (i.e., antibody-dependent cell-mediated cytotoxicity or ADCC), and the deposition of activated complement (i.e., complement-dependent cell-mediated cytotoxicity or CDC). [0004] Due to IdeS's ability to specifically target and cleave IgG, IdeS can significantly reduce antibody-mediated rejection. This is most comprehensively documented in the context of human kidney transplantation, where donor-specific antibodies (DSAs) in transplant recipients recognize epitopes on the donor graft and trigger transplant rejection. Patients with higher levels of DSA can be desensitized by pre-transplant IdeS administration, thereby increasing success rates. Wild-type IdeS has been clinically used to desensitize kidney transplant recipients with higher titers of anti-HLA donor-specific antibodies and may have broader applications in autoimmunity, gene therapy with antibody-rich viral vectors, and to reduce antibody-mediated rejection of xenografts. Brief explanation of the drawing [0031] Figures 1A to 1G show data indicating that IdeS expressed by mammalian cells is active and glycosylated. Figure 1A shows SDSA-PAGE analysis of fractions from NiNTA affinity chromatography of his-tagged IdeS protein. L, load; FT, flow-through. His-tagged IdeS protein was expressed in Escherichia coli (E. coli) cells (EC) or secreted by Expi293F cells (XP). The protein was eluted with PBS containing 250 mM imidazole. The calculated MW of tagged IdeS (excluding glycans) is 36 kD. [0031]Figure 1B shows that the eluate from NiNTA purification was concentrated and separated by SEC. [0031]Figure 1C shows that IdeS (0.25 or 0.5 μg) purified from Expi293F expression medium was treated with PNGase F and analyzed by SDS-PAGE. [0031] Figure 1D shows that cleavage of IgG catalyzed by IdeS generates fragments that can be degraded by non-reducing SDS-PAGE. The first cleavage of the hinge generates Fc/2 and single-cut IgG (scIgG). The second cleavage of the adjacent hinge releases F(ab')2 and two Fc/2 molecules. [0031] Figure 1E shows that the expression medium from Expi293F cells transfected with IdeS was incubated with IVIG, and the cleavage products were detected by SDS-PAGE (upper panel). The expression level of the IdeS protein was evaluated by SDS-PAGE of the expression medium (lower panel). The Asn residue in the consensus N-glycosylation motif (N-X-S/T) was mutated to Gln. [0031] Figure 1F shows that the activity of purified IdeS (20 nM) derived from bacteria was evaluated based on the proteolysis of nonspecific monoclonal IgG1 (20 μM). [0031]Figure 1G shows that the activity of purified IdeS (20 nM) from the Expi293F culture was evaluated based on the proteolysis of nonspecific monoclonal IgG1 (20 μM). [0032] Figures 2A-2C show HLA-II epitopes in IdeS identified by MAPP. Figure 2A shows pulsed treatment of monocyte-derived dendritic cells (DCs) from 10 donors with IdeS. The pMHC-II complex was immunoprecipitated, and the bound IdeS peptide was identified by mass spectrometry. IdeS residues frequently found in the antigen peptide are shown in darker gray. The total number of peptides for each residue across the 10 donors is shown at the bottom. [0032] Figure 2B shows the mapping of the total peptide number of each residue to a model derived from the IdeS crystal structure (PDB 2AVW). Residues that are expressed more frequently in the HLA-II presenting peptide are shown with darker shading. The side chains of the three catalytic residues are labeled and shown as black spheres (201). [0032] Figure 2C shows epitope clusters 1-8 (labeled) on the IdeS structure. The side chains of the three catalytic residues are shown as black spheres (203). Cluster 1 is only partially shown because it is located in the unstructured N-terminus, which is not present in this model. [0033] Figure 3 shows an overview of the predicted and experimentally identified HLA-II epitopes in IdeS and the modifications that reduced immunogenicity. The schematic of