CN-122029191-A - Lysosomal degrading agent based on CTLA-4 and application thereof

Abstract

The present disclosure provides a method for degrading a target protein comprising contacting a T cell with a multispecific binding protein, wherein the multispecific binding protein comprises a) a first cell surface binding moiety that specifically binds to a membrane-bound CTLA-4 on the surface of the T cell, and b) a second binding moiety operably linked to the first cell surface binding moiety and specifically binds to the target protein, wherein binding of the multispecific binding protein to the membrane-bound CTLA-4 on the surface of the T cell promotes internalization of the target protein by the T cell. Following internalization, the multispecific binding protein plus the target protein is transported to a lysosome within the T cell, such that the target protein is capable of degradation within the lysosome. The disclosure also provides multispecific binding protein compositions, host cells for making the multispecific binding protein compositions, and methods of treating diseases using the multispecific binding protein compositions.

Inventors

• E. Kandif
• MUKAI KAORI
• S. Parker
• B nibeier
• V.Z.Sun
• Q.Zhou

Assignees

• 赛诺菲

Dates

Publication Date

20260512

Application Date

20240822

Priority Date

20230823

Claims (20)

1. 1. A method for degrading a target protein, the method comprising: contacting a T cell with a multispecific binding protein, wherein the multispecific binding protein comprises: a) A first cell surface binding moiety that specifically binds to membrane-bound CTLA-4 on the surface of the T cell, and B) A second binding moiety operatively linked to the first cell surface binding moiety and specifically binding to the target protein, Wherein binding of the multispecific binding protein to the membrane-bound CTLA-4 on the surface of the T cell promotes internalization of the target protein by the T cell.
2. 2. The method of claim 1, wherein the target protein degrades in lysosomes after being internalized.
3. 3. The method of claim 1 or 2, wherein the multispecific binding protein expresses increased degradation of the target protein as compared to a reference binding polypeptide.
4. 4. A method according to any one of claims 1-3, wherein the first cell surface binding moiety that specifically binds to membrane-bound CTLA-4 on the surface of the T cell comprises a variable domain of a CTLA-4 antibody or antigen-binding fragment thereof.
5. 5. The method of any one of claims 1-4, wherein the first cell surface binding moiety that specifically binds to membrane-bound CTLA-4 on the surface of the T cell comprises a CTLA-4 binding moiety of a CTLA-4 ligand.
6. 6. The method of claim 5, wherein the CTLA-4 ligand is selected from the group consisting of CD80 and CD 86.
7. 7. The method of claim 6, wherein the CTLA-4 ligand is an extracellular domain of CD80 or CD 86.
8. 8. The method of claim 1, wherein the first cell surface binding moiety comprises a CD80 fragment crystalloid (Fc) fusion polypeptide or a CD86-Fc fusion polypeptide.
9. 9. The method of any one of claims 1-8, wherein the target protein is selected from the group consisting of an antibody, an autoantibody, an inflammatory protein, an interleukin, a cytokine, an interferon, tumor Necrosis Factor (TNF), a growth factor, a hormone, a neurotransmitter, a lipid mediator, an activating factor, an extracellular matrix (ECM) protein, a Wnt protein, a member of the transforming growth factor- β (TGF- β) family, a Notch ligand, and an immune checkpoint protein.
10. 10. The method of any one of claims 1-9, wherein the target protein is a tumor secreting protein.
11. 11. The method of any one of claims 1-10, wherein the target protein is expressed on the membrane of the T cell.
12. 12. The method of claim 11, wherein the T cell line activates T cells or regulatory T (Treg) cells.
13. 13. The method of any one of claims 1-12, wherein the target protein is an immune checkpoint protein.
14. 14. The method of any one of claims 1-13, wherein the target protein is associated with a disease selected from the group consisting of cancer, an autoimmune disease, an inflammatory disorder, an infectious disease, and a neurodegenerative disorder.
15. 15. The method of any one of claims 1-14, wherein the target protein is associated with cancer.
16. 16. The method of any one of claims 1-14, wherein the target protein is associated with an autoimmune disease.
17. 17. The method of any one of claims 1-14, wherein the target protein is associated with an inflammatory disorder.
18. 18. The method of any one of claims 1-17, wherein the first cell surface binding moiety of the multispecific binding protein that specifically binds to membrane-bound CTLA-4 on the surface of the T cell is an antibody or antigen-binding fragment thereof, fc fusion protein, fragment antigen-binding (Fab) fusion, immunoglobulin single variable domain (ISV), or single chain fragment variable domain (scFv).
19. 19. The method of claim 18, wherein the first cell surface binding moiety of the multispecific binding protein that specifically binds to membrane-bound CTLA-4 on the surface of the T cell is an antibody or antigen-binding fragment thereof.
20. 20. The method of claim 18, wherein the first cell surface binding moiety of the multispecific binding protein that specifically binds to membrane-bound CTLA-4 on the surface of the T cell is an Fc fusion protein, wherein the Fc fusion protein comprises a CTLA-4 ligand-Fc fusion protein.

Description

Lysosomal degrading agent based on CTLA-4 and application thereof Priority The present application claims priority from European patent application 23192970.4 filed on 8/23 in 2023 and U.S. Ser. No. 63/674,517 filed on 7/23 in 2024, which are incorporated herein by reference in their entireties. Technical Field The present disclosure relates to novel multispecific binding proteins, or binding fragments thereof, that bind CTLA-4 and a target protein of interest. Methods of using the binding proteins to promote degradation of a target protein of interest are also provided. Sequence listing The present application contains a sequence listing that has been electronically submitted in XML format and incorporated herein by reference in its entirety. The XML file was created at 2024, 2/23, and named 753207_SA9-376-2_ST26.XML, size 55,731 bytes. Background In the last 20 years, target protein degradation technology has attracted great interest in expanding the prospects of pharmaceutically acceptable targets. It also provides a unique mechanism of action for therapeutic agents as "event driven" pharmacology, in contrast to the "occupancy driven" associated with conventional inhibitors. For example, a proteolytic-targeted chimera (PROteolysis TARGETING CHIMERAS) or PROTAC (i.e., a small heterobifunctional molecule that forms a ternary complex with the E3 ubiquitin ligase and target of interest, leading to ubiquitination and degradation of the target) has been advanced through clinical trials. However, the therapeutic potential of PROTAC is hampered by the often poor permeability, pharmacokinetic and pharmacodynamic properties of high molecular weight small molecules (over 1,000 Da). Recently, degradant technologies based on macromolecules such as lysosomal targeting chimeras (LYTAC) have highlighted the potential to utilize macromolecules for targeted degradation of extracellular soluble proteins and membrane-associated proteins. There is a need in the art for targeted degradation strategies, particularly those that degrade targets in specific cell types. Disclosure of Invention The present disclosure is based on the surprising discovery that an endogenous CTLA-4 lysosomal shuttle pathway of T cells can be assigned to degrade target molecules by T cell-specific CTLA-4 mediated lysosomal degradation. The present disclosure provides multispecific binding proteins capable of binding CTLA-4 (e.g., membrane-bound CTLA-4) and a target protein of interest. In certain aspects, binding of the multi-specific binding protein to the membrane-bound CTLA-4 facilitates endocytosis and shuttling of the target protein into the lysosomal compartment of the T cell for subsequent degradation. In certain aspects, the disclosure provides methods for degrading a target protein comprising contacting a T cell with a multispecific binding protein, wherein the multispecific binding protein comprises a) a first cell surface binding moiety that specifically binds to a membrane-bound CTLA-4 on the surface of the T cell, and b) a second binding moiety operably linked to the first cell surface binding moiety and that specifically binds to the target protein, wherein binding of the multispecific binding protein to the membrane-bound CTLA-4 on the surface of the T cell promotes internalization of the target protein by the T cell. In certain aspects, after the target protein is internalized, it degrades in the lysosome. In certain aspects, the multispecific binding protein expresses an increase in degradation of the target protein as compared to a reference binding polypeptide. In certain aspects, the first cell surface binding moiety that specifically binds to a membrane-bound CTLA-4 on the surface of the T cell comprises a variable domain of a CTLA-4 antibody or antigen-binding fragment thereof. In one aspect, the first cell surface binding moiety that specifically binds to membrane-bound CTLA-4 on the surface of the T cell comprises a CTLA-4 binding moiety of a CTLA-4 ligand. In certain aspects, the CTLA-4 ligand is selected from the group consisting of CD80 and CD 86. In certain aspects, the CTLA-4 ligand is an extracellular domain of CD80 or CD 86. In certain aspects, the first cell surface binding moiety comprises a CD80 fragment crystalloid (Fc) fusion polypeptide or a CD86-Fc fusion polypeptide. In certain aspects, the target protein is selected from the group consisting of antibodies, autoantibodies, inflammatory proteins, interleukins, cytokines, interferons, tumor Necrosis Factor (TNF), growth factors, hormones, neurotransmitters, lipid mediators, activation factors, extracellular matrix (ECM) proteins, wnt proteins, members of the transforming growth factor- β (TGF- β) family, notch ligands, and immune checkpoint proteins. In certain aspects, the target protein is a tumor secreting protein. In certain aspects, the target protein is expressed on the membrane of the T cell. In one aspect, the T cell is an activated T cell or a regulatory T