CTLA-4-based lysosome degradation agent and application thereof

Abstract

The present disclosure provides 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 binding 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 binding to the target protein, wherein binding of the multispecific binding protein to the membrane-binding CTLA-4 on the surface of the T cell promotes internalization of the target protein by the T cell. After internalization, the multispecific binding protein plus the target protein is transported to the lysosome within the T cell, such that the target protein can be degraded within the lysosome. The disclosure also provides multispecific binding protein compositions, host cells for making the multispecific binding protein compositions, and methods of using the multispecific binding protein compositions in the treatment of disease.

Description

priority

[0001] This application claims priority to European Patent Application No. 23192970.4, filed August 23, 2023, and U.S. Serial No. 63 / 674,517, filed July 23, 2024, which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates to novel multispecific binding proteins or binding fragments thereof that bind to CTLA-4 and target proteins. A method for promoting the degradation of target proteins using said binding proteins is also provided. sequence list

[0003] This application contains a list of sequences that have been electronically submitted in XML format and incorporated herein by reference in their entirety. The XML file, created on February 23, 2024, is named 753207_SA9-376-2_ST26.xml and is 55,731 bytes in size. Background Technology

[0004] Over the past two decades, target protein degradation technologies have generated significant interest in expanding the prospects for druggable targets. They also offer a unique mechanism of action for therapeutic agents as “event-driven” pharmacology, entirely different from the “occupancy-driven” approach associated with conventional inhibitors. For example, PROteolysis TArgeting Chimeras (PROTACs), or small heterobifunctional molecules that form ternary complexes with E3 ubiquitin ligases and the target, leading to ubiquitination and degradation of the target, have made progress in clinical trials. However, the therapeutic potential of PROTACs is hampered by the poor permeability, pharmacokinetic, and pharmacodynamic properties commonly associated with high molecular weight small molecules (over 1,000 Da). Recently, degradative agent technologies based on macromolecules such as lysosome-targeting chimeras (LYTACs) have highlighted the potential for targeted degradation of extracellular soluble proteins and membrane-associated proteins using macromolecules. Targeted degradation strategies, particularly those capable of degrading targets in specific cell types, are needed in this field. Summary of the Invention

[0005] This disclosure is based on the surprising discovery that the endogenous CTLA-4 lysosomal shuttle pathway of T cells can be assigned to degrade target molecules via T cell-specific CTLA-4-mediated lysosomal degradation. This specification provides multispecific binding proteins capable of binding CTLA-4 (e.g., membrane-bound CTLA-4) to target proteins. In some respects, the binding of the multispecific binding protein to membrane-bound CTLA-4 promotes the endocytosis and shuttle of the target protein into the lysosomal compartment of the T cell for subsequent degradation.

[0006] In some aspects, this disclosure provides 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 portion that specifically binds to a membrane-bound CTLA-4 on the surface of the T cell; and b) a second binding portion operatively connected to the first cell surface binding portion and specifically binding to the target protein, wherein the binding of the multispecific binding protein to the membrane-bound CTLA-4 on the surface of the T cell promotes the internalization of the target protein by the T cell.

[0007] In some respects, the target protein is degraded in lysosomes after internalization. In other respects, the multispecific binding protein expresses increased degradation of the target protein compared to a reference binding peptide.

[0008] In some aspects, the first cell surface binding portion 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 an antigen-binding fragment thereof. In another aspect, the first cell surface binding portion that specifically binds to membrane-bound CTLA-4 on the surface of the T cell comprises a CTLA-4 binding portion of a CTLA-4 ligand. In some aspects, the CTLA-4 ligand is selected from the group consisting of CD80 and CD86. In some aspects, the CTLA-4 ligand is an extracellular domain of CD80 or CD86. In some aspects, the first cell surface binding portion comprises a CD80 fragment crystallizable (Fc) fusion polypeptide or a CD86-Fc fusion polypeptide.

[0009] In some respects, 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, activating factors, extracellular matrix (ECM) proteins, Wnt proteins, members of the transforming growth factor-β (TGF-β) family, Notch ligands, and immune checkpoint proteins.

[0010] In some respects, the target protein is a tumor-secreting protein. In some respects, the target protein is expressed on the membrane of the T cell. In some respects, the T cell is an activated T cell or a regulatory T (Treg) cell. In some respects, the target protein is an immune checkpoint protein. In some respects, the target protein is associated with diseases selected from the group consisting of: cancer, autoimmune diseases, inflammatory disorders, infectious diseases, and neurodegenerative disorders. In some respects, the target protein is associated with cancer. In some respects, the target protein is associated with autoimmune diseases. In some respects, the target protein is associated with inflammatory disorders.

[0011] In some respects, the first cell surface binding portion of the multispecific binding protein that specifically binds to the membrane-bound CTLA-4 on the surface of the T cell is an antibody or its antigen-binding fragment, an Fc fusion protein, a fragment antigen-binding (Fab) fusion, an immunoglobulin single variable domain (ISV), or a single-chain fragment variable domain (scFv).

[0012] In some respects, the first cell surface binding portion of the multispecific binding protein that specifically binds to CTLA-4 on the surface of the T cell is an antibody or its antigen-binding fragment.

[0013] In some respects, the first cell surface binding portion of the multispecific binding protein that specifically binds to the 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.

[0014] In some respects, the first cell surface binding portion of the multispecific binding protein that specifically binds to the membrane-bound CTLA-4 on the surface of the T cell is an scFv, wherein the scFv is a linear scFv or a tandem scFv.

[0015] In some respects, the first cell surface binding portion of the multispecific binding protein that specifically binds to the membrane-bound CTLA-4 on the surface of the T cell is an ISV.

[0016] In some respects, the ISV is V HH Humanized V HH Or camel-like V H .

[0017] In some respects, the second binding portion of the multispecific binding protein that specifically binds to the target protein is selected from the group consisting of: antigen, antibody or antigen-binding fragment thereof, autoantibody, Fc fusion protein, Fab, scFv, ISV, AFFIBODY® or peptide.

[0018] In some respects, the second binding portion of the multispecific binding protein that specifically binds to the target protein is an antibody or its antigen-binding fragment.

[0019] In some aspects, the second binding portion of the multispecific binding protein that specifically binds to the target protein is an ISV. In some aspects, the ISV is a V. HH Humanized V HH Or camel-like V H .

[0020] In some aspects, the first cell surface binding portion of the multispecific binding protein exhibits pH-dependent binding to the membrane-bound CTLA-4 on the surface of the T cell. In some aspects, the second binding portion of the multispecific binding protein exhibits pH-dependent binding to the target protein. In some aspects, the binding of the multispecific binding protein decreases at acidic pH conditions.

[0021] In some aspects, the first cell surface binding portion of the multispecific binding protein binds to membrane-bound CTLA-4 on the surface of the T cell with an affinity from about 100 pM to about 1 µM. In some aspects, the second binding portion of the multispecific binding protein binds to the target protein with an affinity from about 100 pM to about 1 µM.

[0022] In some respects, the multispecific binding protein contains one or more mutations or glycan modifications to modulate the Fc-mediated effector function.

[0023] In some respects, the multispecific binding protein contains one or more mutations to regulate its half-life.

[0024] In one aspect, this disclosure provides a multispecific binding protein comprising: a) a first cell surface binding portion that specifically binds to membrane-bound CTLA-4 on the surface of a T cell; and b) a second binding portion operatively connected to the first cell surface binding portion and specifically binding to the target protein, such that the multispecific binding protein binds to the membrane-bound CTLA-4 and the target protein on the surface of the T cell, wherein binding to the membrane-bound CTLA-4 on the surface of the T cell promotes the internalization of the target protein by the T cell.

[0025] In some respects, the target protein is degraded in lysosomes after internalization. In other respects, the multispecific binding protein exhibits increased degradation of the target protein compared to a reference binding peptide.

[0026] In some respects, the first cell surface binding portion that specifically binds to membrane-bound CTLA-4 on the surface of the T cells is a CTLA-4 ligand. In some respects, the CTLA-4 ligand is selected from the group consisting of CD80 and CD86.

[0027] In some respects, 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, activating factors, extracellular matrix (ECM) proteins, Wnt proteins, members of the transforming growth factor-β (TGF-β) family, Notch ligands, and immune checkpoint proteins.

[0028] In some aspects, the target protein is a tumor-secreting protein. In some aspects, the target protein is expressed on the membrane of a T cell. In some aspects, the T cell is an activated T cell or a Treg cell. In some aspects, the target protein is an immune checkpoint protein. In some aspects, the target protein is associated with diseases selected from the group consisting of: cancer, autoimmune diseases, inflammatory disorders, infectious diseases, and neurodegenerative disorders. In some aspects, the target protein is associated with cancer. In some aspects, the target protein is associated with autoimmune diseases. In some aspects, the target protein is associated with inflammatory disorders.

[0029] In some respects, the first cell surface binding portion of the multispecific binding protein that specifically binds to the membrane on the surface of the T cell, CTLA-4, is an antibody or its antigen-binding fragment, an Fc fusion protein, a Fab fusion, an ISV, or a scFv.

[0030] In some respects, the first cell surface binding portion that specifically binds to CTLA-4 on the surface of the T cell is an antibody or its antigen-binding fragment.

[0031] In some respects, the first cell surface binding portion that specifically binds to 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.

[0032] In some aspects, the first cell surface binding portion of the multispecific binding protein that specifically binds to CTLA-4 on the surface of the T cell is an scFv, wherein the scFv is a linear scFv or a tandem scFv. In some aspects, the first cell surface binding portion of the multispecific binding protein that specifically binds to CTLA-4 on the surface of the T cell is an ISV. In some aspects, the ISV is a V HH Humanized V HH Or camel-like V H .

[0033] In some aspects, the second binding moiety that specifically binds to the target protein is selected from the group consisting of: antigen, antibody or antigen-binding fragment thereof, autoantibody, Fc fusion, Fab, scFv, ISV, AFFIBODY®, or peptide. In some aspects, the second binding moiety that specifically binds to the target protein is an ISV. In some aspects, the ISV is V. HH Humanized V HH Or camel-like V H .

[0034] In some aspects, the first cell surface binding portion of the multispecific binding protein exhibits pH-dependent binding to membrane-bound CTLA-4 on the surface of the T cell. In some aspects, the second binding portion of the multispecific binding protein exhibits pH-dependent binding to the target protein. In some aspects, the multispecific binding protein further comprises modifications that allow the multispecific binding protein to bind to membrane-bound CTLA-4 on the surface of the T cell in a pH-dependent manner. In some aspects, the modifications reduce binding at acidic pH.

[0035] In some aspects, the first cell surface binding portion of the multispecific binding protein binds to the membrane-bound CTLA-4 on the surface of the T cell with an affinity from about 100 pM to about 1 µM. In some aspects, the second binding portion of the multispecific binding protein binds to the target protein with an affinity from about 100 pM to about 1 µM.

[0036] In some respects, the multispecific binding protein further comprises one or more mutations or glycan modifications to modulate the Fc-mediated effector function.

[0037] In some respects, the multispecific binding protein further includes one or more mutations to regulate its half-life.

[0038] In one aspect, this disclosure provides pharmaceutical compositions comprising any of the multispecific binding proteins disclosed herein. In another aspect, the compositions further comprise a pharmaceutically acceptable carrier.

[0039] In one aspect, this disclosure provides isolated nucleic acid molecules encoding any of the multispecific binding proteins disclosed herein. In some aspects, this disclosure provides expression vectors comprising nucleic acid molecules as disclosed herein. In some aspects, this disclosure provides host cells comprising expression vectors as disclosed herein.

[0040] In one aspect, this disclosure provides a method for treating a subject suffering from a disease associated with a target protein or a soluble target protein, the method comprising administering to the subject a therapeutically effective amount of any multispecific binding protein described herein or any pharmaceutical composition disclosed herein.

[0041] In one aspect, this disclosure provides a method of treating a subject, wherein the binding of the multispecific binding protein to the membrane-bound CTLA-4 on the surface of the T cell promotes the internalization of the target protein by the T cell and the transport of the target protein by the T cell to the lysosome within the T cell, so that the target protein can be degraded in the lysosome, thereby treating the disease of the subject.

[0042] In some respects, the disease is selected from the group consisting of: cancer, autoimmune diseases, inflammatory disorders, infectious diseases, and neurodegenerative disorders. In some respects, the disease is cancer. In some respects, the disease is an autoimmune disease. In some respects, the disease is an inflammatory disorder.

[0043] In some respects, this disclosure provides a method of treating a subject, wherein the multispecific binding protein is administered via intravenous, subcutaneous, intramuscular, or intradermal injection.

[0044] The above description of this disclosure is non-limiting, and other features and advantages of the disclosed multispecific binding proteins or their binding fragments or derivatives, as well as the methods, will become clear from the following description of the drawings, detailed description, and claims. [Attached image description]

[0045] Figure 1 This illustration shows a schematic diagram of the internalization of target proteins (such as TNFα) on the surface of CTLA-4 cells by the TNFα / CTLA-4 multispecific binding protein disclosed in this paper. Wild-type Raji cell lines (Raji-empty cells) were engineered to stably express human CTLA-4 on the cell surface membrane (Raji-CTLA-4 cells).

[0046] Figures 2A-2B This demonstrates the effect of adding anti-CTLA-4 antibody ( Figure 2A After ) or after adding antibody isotype control ( Figure 2B One hour later, Raji-CTLA-4 cells internalized CTLA-4. As confirmed by confocal microscopy, CTLA-4 was rapidly internalized after one hour of incubation with anti-CTLA-4 antibody in cells expressing Raji-CTLA-4. Figure 2A ).

[0047] Figures 3A-3B This demonstrates the effect of adding anti-CTLA-4 antibody ( Figure 3A After ) or after adding antibody isotype control ( Figure 3B Four hours later, Raji-CTLA-4 cells internalized CTLA-4.

[0048] Figure 4 This is a schematic diagram of the bispecific antibody complex, used to visualize the internalization and lysosomal transport steps of the target protein TNFα after binding to the anti-TNFα / CTLA-4 bispecific antibody construct disclosed herein. The bispecific antibody complex consists of biotinylated human TNFα, streptavidin conjugated to AlexaFlour488 (streptavidin-AF488), and the anti-TNFα / CTLA-4 bispecific antibody construct described herein.

[0049] Figure 5 To combine Raji-CTLA-4 cells with Figure 4 Representative confocal maximum intensity projection images of the bispecific antibody complexes described above after incubation for 2 hours (left column) or 4 hours (right column). After incubation, cells were fixed and co-stained with fluorescently labeled endosome and lysosomal markers, namely anti-EEA1 and anti-LAMP1 antibodies, and nuclear staining agent (Hoechst). Scale bar: 15 μm.

[0050] Figure 6 To combine Raji-CTLA-4 cells with Figure 4 Representative confocal images of the bispecific antibody complexes described herein incubated together for 1 hour (top), 2 hours (middle), or 4 hours (bottom). Scale bar: 20 μm.

[0051] Figure 7 To combine Raji-CTLA-4 cells with Figure 4 Representative confocal maximum intensity projection images of the bispecific antibody complexes described above, incubated together for 1 hour (left column), 2 hours (middle column), or 4 hours (right column). After incubation, cells were fixed and co-stained with endosome and lysosomal markers, namely rabbit anti-EEA1 and mouse anti-LAMP1 antibodies, fluorescently labeled goat anti-rabbit and goat anti-mouse secondary antibodies, and nuclear staining agent (Hoechst). Images were captured using ZEISS Airyscan Joint Deconvolution for optimized resolution and channel separation. Scale bar: 15 μm.

[0052] Figure 8 In order to utilize and such Figure 7 Representative confocal maximum intensity projection superimposed images of Raji-CTLA-4 cells incubated for 1 hour (top), 2 hours (middle), or 4 hours (bottom) using the same experimental setup described above. The superimposed images show the fluorescent labeling of the bispecific antibody complexes EEA1, LAMP1, and the nucleus. Scale bar: 5 μm.

[0053] Figure 9 In order to be in Figure 7 and Figure 8 The plot shows the weighted colocalization values ​​of different colocalization levels of EEA1 and LAMP1 fluorescent markers quantified by staining from the bispecific antibody complex during the 1-hour, 2-hour, and 4-hour time processes described in the experiment.

[0054] Figure 10 Showing Figure 4Two schematic diagrams of the bispecific antibody complex described herein, which is further modified by conjugating pHrodo-red to a bispecific antibody (top) or streptavidin-AF488 (bottom).

[0055] Figure 11 Representative confocal maximum intensity projection overlay images of Raji-CTLA-4 cells after incubation with pHrodo-red staining agent and LysoView™ staining agent (LysoView™ is a lysosomal marker) for 0, 9, 30, or 45 minutes are shown.

[0056] Figure 12 Two figures are shown, illustrating the effect of combining Raji-CTLA-4 cells with... Figure 10 The modified bispecific antibody conjugated to pHrodo-streptavidin described in the article or Figure 4 The average fluorescence intensity (MFI) of images collected during a 60-minute time process generated by incubating the pHodo antibody complex described herein together.

[0057] Figure 13 The following figure shows an MFI of images collected during a 60-minute time process, generated by incubating Raji-empty cells or Raji-CTLA-4 cells with a modified bispecific antibody conjugated to pHrodo-red.

[0058] Figures 14A-14B This study demonstrates the internalization of TNFα in Raji-CTLA4 cells or control cells using a TNF / CTLA-4 bispecific antibody. Figure 14A The image shows CTLA-4 staining in Raji-CTLA4 cells and a control (Raji-empty cells). Figure 14B The study demonstrated the internalization of TNFα in Raji-CTLA-4 cells after treatment with 25 or 100 nM anti-TNFα / CTLA-4 bispecific antibody constructs plus fluorescently labeled TNFα at concentrations of 25 and 100 nM, or with antibody isotype controls plus fluorescently labeled TNFα.

[0059] Figure 15This study demonstrates the degradation of TNFα by the anti-TNFα / CTLA-4 bispecific antibody, as determined by Western blotting. Raji-CTLA-4 cells or Raji-empty cells (control) were incubated for 2 hours with the anti-TNFα / CTLA-4 bispecific antibody plus TNFα, or with an antibody isotype control plus TNFα, followed by washing. The two cell sample groups were then treated with bafilomycin (a potent lysosomal inhibitor) or DMSO at several time points.

[0060] Figures 16A-16B FACS analysis demonstrated the internalization of TNFα via the anti-TNFα / CTLA-4 bispecific antibody. Figure 16A A representative FACS image is shown, displaying CD4 in a peripheral blood mononuclear cell (PBMC) sample. + Cell population. Figure 16B This study demonstrates the internalization of fluorescently labeled TNFα in phytohemagglutinin (PHA)-activated PBMCs after 4 hours and 24 hours of incubation using either an anti-TNFα / CTLA-4 bispecific antibody or an antibody isotype control. The PHA-activated PBMCs were derived from two donors (“Donor 1” and “Donor 2”).

[0061] Figure 17 Demonstrated the use of three different anti-TNFα / CTLA-4 bispecific V HH The construct, after incubation with fluorescently labeled TNFα at increasing concentrations, resulted in TNFα internalization in Raji-CTLA-4 cells or Raji-empty cells (control). In Raji-empty cells or in V cells utilizing a single binding domain expressing CTLA-4 or TNFα... HH No TNFα internalization was observed in the construct control.

[0062] Figure 18 for Figure 17 The results of the FACs analysis performed are graphically represented as MFI.

[0063] Figure 19 Demonstrates bispecificity with fluorescently labeled TNFα and anti-TNFα / CTLA-4 VHH Constructs or controls expressing a single binding domain of CTLA-4 or TNFα HH After co-incubation with the constructs, the fold increase in TNFα internalization in Raji-CTLA-4 cells relative to Raji-empty cells (control) was observed.

[0064] Figure 20 Demonstrated the effectiveness of using TNFα plus three anti-TNFα / CTLA-4 bispecific V HHWestern blot images of TNFα degradation in lysosomes of Raji-CTLA-4 cells after incubation for 2, 9, or 24 hours with one of the constructs. As a control, bafloxacin was added to some samples.

[0065] Figure 21 This image shows Western blot images of decreased TNFα levels in the cell culture medium after adding TNFα (at 12.5 nM or 50 nM) to Raji-CTLA-4 cells with 25 nM or 100 nM of three anti-TNFα / CTLA-4 bispecific V-type inhibitors. HH Constructor or V HH The control group was incubated together for 24 hours. Detailed Implementation

[0066] This document provides compositions and methods for degrading target molecules via CTLA-4-mediated lysosomal degradation. The compositions and methods disclosed herein utilize a multispecific binding protein comprising a first binding portion that specifically interacts with CTLA-4 on T cells (e.g., membrane-bound CTLA-4) and a second binding portion that specifically binds to a target molecule (e.g., a pathogenic protein).

[0067] It should be understood that the methods described in this disclosure are not limited to specific methods and experimental conditions, and that methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Peptides and isolated peptides

[0068] The term "peptide" refers to any polymeric amino acid chain and encompasses natural or artificial proteins, peptide analogs or variants of protein sequences, or fragments thereof, unless otherwise contradicted by the context. Peptides can be monomeric or polymeric. For a peptide (e.g., a peptide encoding a first cell surface binding portion that specifically binds to CTLA-4 on the surface of CTLA-4-positive cells and / or a second binding portion operatively linked to the first cell surface binding portion and specifically binding to the target protein), a fragment of the peptide optionally contains at least one continuous or non-linear epitope of the peptide. The fragment peptide can be about 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, or more amino acids in length while retaining the ability to bind to both CTLA-4 and the target protein. The precise boundaries of at least one epitope fragment can be verified by those skilled in the art. The polypeptide fragment contains, for example, at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, or at least about 20 consecutive amino acids, at least about 50 consecutive amino acids, at least about 100 consecutive amino acids, at least about 150 consecutive amino acids, at least about 200 consecutive amino acids, at least about 250 consecutive amino acids, at least about 300 consecutive amino acids, or at least about 400 consecutive amino acids.

[0069] In some respects, the first cell surface binding portion and the second binding portion of the polypeptide are "isolated polypeptides". The term "isolated polypeptide" refers to a protein or polypeptide that, due to its origin or derivative, is not associated with its naturally occurring associated components in its native state; and is substantially free of other proteins from the same species. Isolated recombinant polypeptides are expressed by cells from different species. In some respects, isolated polypeptides are not naturally occurring. Chemically synthesized or synthesized in a cellular system, a protein or polypeptide may differ from the cell of its natural origin and thus be "isolated" from its naturally occurring associated components. Proteins or polypeptides can also be isolated to be substantially free of their naturally occurring associated components using protein purification techniques. Binding proteins or binding peptides

[0070] As used herein, the terms “binding protein,” “binding polypeptide,” or “multispecific binding polypeptide or protein” refer to a protein or polypeptide (e.g., an antibody or its specific binding fragment, Fab, scFv, immunoglobulin with a single variable domain such as the NANOBODY® molecule, AFFIBODY®, V). HHA binding protein or peptide contains at least one binding site responsible for selectively binding to a target antigen (e.g., a human antigen). The binding site may include an antibody variable domain, a receptor ligand binding site, or a ligand receptor binding site. In some respects, the binding protein or peptide contains multiple (e.g., two, three, four, or more) binding sites. In some respects, the binding protein or peptide is not a therapeutic enzyme.

[0071] Affinity antibodies (or non-immunoglobulin-binding scaffold proteins) refer to any class of small (approximately 6 kDa) polypeptide antibody mimics containing a triple α-helix bundle domain of approximately 58 amino acids in length. Affinity antibodies are derived from the immunoglobulin-binding domain of staphylococcal protein A. See, for example, Nord et al., Protein Eng. 8:601-608 (1995). Affinity antibodies are highly stable, withstanding temperatures up to 90°C, and lack Fc function. Affinity antibody binding sites can be synthesized by mutagenesis of staphylococcal protein A-related proteins (e.g., protein Z) derived from a domain of protein A (e.g., domain B) and selection of mutant polypeptides with binding affinity for the target. Affinity antibody binding sites can also be generated by methods described in U.S. Patent Nos. 6,740,734, 6,602,977, and WO 2000 / 063243. Ligands and antigens

[0072] The term "ligand" refers to any substance that can bind to or be bound by another substance. As used herein, the term "antigen" or "target antigen" refers to a molecule or part of a molecule that can be bound to a binding site of a polypeptide, for example, any substance that can generate an antibody. A target antigen may have one or more epitopes.

[0073] Although the term "antigen" is commonly used when referring to antibody-binding substrates and "ligand" is often used when referring to receptor-binding substrates, the terms are interchangeable and cover a wide range of overlapping chemical entities. For the avoidance of ambiguity, antigen and ligand are used interchangeably throughout this document.

[0074] The antigen / ligand implementation scheme can be peptide, polypeptide, protein, aptamer, polysaccharide, sugar molecule, carbohydrate, lipid, oligonucleotide, polynucleotide, synthetic molecule, inorganic molecule, organic molecule, and any combination thereof. Immunoglobulin domain

[0075] As used herein, the term immunoglobulin domain may refer to immunoglobulin A, immunoglobulin D, immunoglobulin E, immunoglobulin G, or immunoglobulin M. An immunoglobulin domain may be a region of the immunoglobulin heavy chain or a fragment thereof. In some cases, the immunoglobulin domain originates from an antibody (e.g., monoclonal antibody, mammalian antibody, recombinant antibody, chimeric antibody, engineered antibody, human antibody, humanized antibody) or an antigen-binding fragment thereof. Antibody

[0076] As used herein, the term "antibody" refers to an assembly (e.g., a complete antibody molecule, its antibody fragment, or a variant) that exhibits significant, known specific immunoreactivity against a target antigen (e.g., cell surface CTLA-4 on T cells or a target protein-associated antigen). Antibodies and immunoglobulins comprise light and heavy chains, with or without interchain covalent linkages. The basic immunoglobulin structures of vertebrate systems are relatively well understood.

[0077] As will be discussed in more detail below, the general term "antibody" includes five distinct classes of antibodies that can be distinguished biochemically. While all five classes of antibodies are clearly within the scope of this disclosure, the following discussion will generally refer to the IgG class of immunoglobulin molecules. Regarding IgG, immunoglobulins consist of two identical light chains with a molecular weight of approximately 23,000 Daltons and two identical heavy chains with molecular weights of 53,000–70,000. The four chains are linked by disulfide bonds in a "Y" configuration, where the light chain begins at the opening of the "Y" and continues to surround the heavy chain throughout the variable region.

[0078] Immunoglobulin light chains are classified as κ or λ (kappa or lambda). Each heavy chain class can bind to a κ or λ light chain. Typically, light and heavy chains are covalently bonded to each other, and when immunoglobulins are generated by hybridomas, B cells, or genetically engineered host cells, the “tail” portions of the two heavy chains are bonded to each other via covalent disulfide linkages or non-covalent linkages. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration to the C-terminus at the bottom of each chain. Those skilled in the art will understand that heavy chains are classified as γ, μ, α, δ, or ε (gamma, mu, alpha, delta, or epsilon), among which there are several subclasses (e.g., γ1-γ4). The nature of this chain determines the “class” of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) confer functional specialization. The modifications of each of the categories and types are readily discernible to a person skilled in the art in consideration of this disclosure, and are therefore within the scope of this disclosure.

[0079] Both light and heavy chains are categorized into regions that share structural and functional homology. The term "region" refers to a part or portion of an immunoglobulin or antibody chain and includes constant or variable regions, as well as more discrete portions of said regions. For example, variable regions of light chains include "complementarity-determining regions" or "CDRs" scattered within "frame regions" or "FRs" as defined herein. Constant and variable structural domains

[0080] Immunoglobulin heavy or light chain regions can be defined as "constant" (C) regions or "variable" (V) regions, based on whether, in the case of a "constant region," there is a relative lack of sequence variation within the region of each member, or in the case of a "variable region," there is significant variation within the region of each member. The terms "constant region" and "variable region" can also be used functionally. In this regard, it should be understood that the variable regions of immunoglobulins or antibodies determine antigen recognition and specificity. Conversely, the constant regions of immunoglobulins or antibodies confer important effector functions, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. The subunit structures and three-dimensional conformations of the constant regions of different immunoglobulin classes are well known.

[0081] The constant and variable regions of the immunoglobulin heavy and light chains fold into domains. The term "domain" refers to a globular region of a heavy or light chain containing a peptide ring (e.g., containing 3 to 4 peptide rings), which is stabilized, for example, by a β-sheet and / or intrachain disulfide bond. Constant region domains on the light chain of an immunoglobulin are interchangeably referred to as "light chain constant region domains," "CL regions," or "CL domains." Constant domains on the heavy chain (e.g., hinge, CH1, CH2, or CH3 domains) are interchangeably referred to as "heavy chain constant region domains," "CH" region domains, or "CH domains." Variable domains on the light chain are interchangeably referred to as "light chain variable region domains," "VL region domains," or "VL domains." "Variable domains" on the heavy chain are interchangeably referred to as "heavy chain variable region domains," "VH region domains," or "VH domains."

[0082] By convention, the variable constant region domains are numbered as they move further away from the antigen-binding site or N-terminus of the immunoglobulin or antibody. The N-terminus of each immunoglobulin heavy and light chain is the variable region, and the C-terminus is the constant region; the CH3 and CL domains actually contain the C-termini of the heavy and light chains, respectively. Therefore, the domains of light chain immunoglobulins are aligned in the VL-CL direction, while the domains of heavy chains are aligned in the VH-CH1-hinge-CH2-CH3 direction.

[0083] The amino acid assignments for each variable domain are based on the definitions in Kabat, Sequence of Immunologically Targeted Proteins (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chain variable regions or between different light chain variable regions are assigned the same number. The CDRs 1, 2, and 3 of the VL domain are also referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively. The CDRs 1, 2, and 3 of the VH domain are also referred to herein as CDR-H1, CDR-H2, and CDR-H3, respectively. If so, the CDR assignments may be based on IMGT® (Lefranc et al., Developmental and Comparative Immunology 27:55-77; 2003) rather than Kabat. The heavy chain constant regions were numbered using the EU index as described in Kabat (Kabat, Sequence of Immunologically Targeted Proteins, National Institutes of Health, Bethesda, MD, 1987 and 1991).

[0084] In some aspects, the multispecific binding protein comprises: a) a first cell surface binding portion that specifically binds to membrane-bound CTLA-4 on the surface of a T cell; and b) a second binding portion operatively linked to the first cell surface binding portion and specifically binding to the target protein, such that the multispecific binding protein binds to both the membrane-bound CTLA-4 and the target protein on the surface of the T cell, wherein binding to the membrane-bound CTLA-4 on the surface of the T cell promotes the internalization of the target protein by the T cell. In some aspects, after internalization, the target protein is degraded in lysosomes. In some aspects, the multispecific binding protein exhibits increased degradation of the target protein compared to a reference binding peptide.

[0085] In some aspects, the first cell surface binding portion that specifically binds to the membrane-bound CTLA-4 on the surface of the T cell is a CTLA-4 ligand. In some aspects, the CTLA-4 ligand is selected from the group consisting of CD80 and CD86. In some aspects, the first cell surface binding portion that specifically binds to the membrane-bound CTLA-4 on the surface of the T cell is an antibody or an antigen-binding fragment thereof. In some aspects, the first cell surface binding portion that specifically binds to the 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.

[0086] In some respects, the second binding moiety that specifically binds to the target protein is selected from the group consisting of: antigen, antibody or antigen-binding fragment thereof, autoantibody, Fc fusion, Fab, scFv, ISVD, NANOBODY®, AFFIBODY® or peptide.

[0087] Unbound by any specific mechanistic theory, upon binding to membrane-bound CTLA-4, multispecific binding proteins promote the internalization (e.g., endocytosis) of target proteins into T cells, initiating a series of intracellular events that ultimately lead to the shuttle or transport of the target protein to the lysosomal compartment of the T cell for subsequent degradation. By utilizing or assigning the endogenous CTLA-4 shuttle pathway of T cells, the methods disclosed herein enable the efficient and selective degradation of a variety of target molecules (soluble and membrane-bound), such as tumor-secreted proteins, autoantibodies, inflammatory proteins, interleukins, and signaling molecules. This can be used in targeted therapies to precisely control the degradation of specific molecules, providing a pathway for treating diseases such as autoimmune disorders, cancer, and inflammatory conditions, resulting in a more balanced immune response and improved clinical outcomes.

[0088] As used herein, the term “cytotoxic T-lymphocyte antigen 4” or “CTLA-4” refers to a membrane-bound receptor, a member of the immunoglobulin (Ig) superfamily, expressed by activated T cells. CTLA-4 is homologous to the T cell costimulatory protein CD28 and binds to B7-1 / CD80 and B7-2 / CD86 ligands present on antigen-presenting cells (APCs). CTLA-4 is also found on the surface membrane of regulatory T cells. CTLA-4 is also known as cytotoxic T-lymphocyte-associated protein 4, CD152, insulin-dependent diabetes mellitus 12 (IDDM12), celiac 3 (CELIAC3), GRD4, and GSE. The term “CTLA-4” includes any variant or isotype of CTLA-4 naturally expressed by cells.

[0089] As used herein, the term "T cell" is defined as a thymus-derived lymphocyte involved in a variety of cell-mediated immune responses. In some respects, the T cells referred to are regulatory T cells. As used herein, the term "regulatory T cell" refers to a CD4+CD25+FoxP3+ T cell with suppressive properties. "Treg" is an abbreviation used herein for regulatory T cells. T cells, activated T cells, and Treg cells can express membrane-bound CTLA-4.

[0090] The terms "specific binding," "specifically binds to," and "specific to" refer to the formation of a relatively stable complex under physiological conditions by a multispecifically binding protein, its binding fragment, or derivative with an antigen. Specific binding can occur through at least approximately 1 × 10⁻⁶ −6Characterized by M or a smaller equilibrium dissociation constant (e.g., K) D The smaller the value, the tighter the binding. Methods for determining whether two molecules are specifically bound include, for example, equilibrium dialysis and surface plasmon resonance.

[0091] In some respects, the constant domain can be an Fc domain. As used herein, the term "Fc domain" or "Fc region" (which may be used interchangeably) is defined as part of a heavy chain constant region that begins in the hinge region, just upstream of the papain cleavage site (i.e., residue 216 in IgG, taking the first residue of the heavy chain constant region as 114), and terminates at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

[0092] The terms “Fc variant,” “modified Fc,” and “engineered Fc,” which may be used interchangeably herein, refer to molecules or sequences modified from natural Fc but still containing a binding site for the rescue receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with the rescue receptor are known in the art. Modified Fc domains may comprise molecules or sequences humanized from nonhuman natural Fc. Furthermore, natural Fc contains regions that can be removed because they provide structural features or biological activities not desired by the conjugated composition. Thus, the term “modified Fc domain” includes molecules or sequences lacking one or more natural Fc sites or residues or one or more Fc sites or residues that have been modified to affect or participate in: (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity when expressed in selected host cells, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the rescue receptor, or (7) antibody-dependent cytotoxicity (ADCC).

[0093] As used herein, "effective function" refers to a biochemical event resulting from the interaction between the antibody Fc region and an Fc receptor or ligand. A "functional Fc region" possesses the "effective function" of a native Fc region. Exemplary "effective functions" include antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP).

[0094] The term “EU index” refers to the EU numbering convention for antibody constant regions, as described in Edelman, GM. et al., Proceedings of the National Academy of Sciences, 63, 78-85 (1969) and Kabat et al., Sequences of Immunologically Objective Proteins, U.S. Department of Health and Human Services, 5th Edition, 1991, each incorporated herein by reference in its entirety. Unless otherwise stated, all antibody Fc region numbers used herein correspond to the EU numbering scheme as described in Edelman et al. (Proceedings of the National Academy of Sciences 63(1): 78-85. 1969).

[0095] Furthermore, without being bound by any specific mechanistic theory, when the first binding portion of the multispecific binding protein binds to CTLA-4 on the surface of T cells, this triggers receptor-mediated endocytosis and internalization of the complex, which contains the CTLA-4 receptor and the multispecific binding protein that binds to CTLA-4 via the first binding portion and to the target via the second binding portion. Specifically, the interaction of the first binding portion with CTLA-4 causes the aggregation of the CTLA-4 receptor, leading to the formation of endocytic vesicles. This aggregation induces the recruitment of adaptors and clathrin-coated pits to the cell membrane, initiating receptor-mediated endocytosis. After the formation of clathrin-coated pits, the aggregated CTLA-4 receptors are internalized through invagination of the plasma membrane, resulting in the formation of clathrin-coated vesicles. These vesicles containing the CTLA-4 receptor detach from the plasma membrane and enter the cytoplasm. Once inside the cytoplasm, the clathrin-coated vesicles shed their clathrin coating, forming uncoated endocytic vesicles that transport the internalized CTLA-4 receptors to early endosomes. Early endosomes are intracellular compartments involved in the sorting and transport of cargo molecules. Within early endosomes, the internalized CTLA-4 receptor can undergo further sorting. The CTLA-4 receptor moves from early endosomes to late endosomes, characterized by a lower pH environment due to the presence of proton pumps. The gradually decreasing pH in late endosomes triggers the fusion of lysosomes (specialized organelles containing various hydrolases) with the late endosome. This fusion event leads to the formation of endolysosomes. The target protein, or a target protein bound to a multispecific binding protein or its binding fragment or derivative, is co-internalized with CTLA-4 into the endolysosomal compartment. The endolysosome then fuses with the lysosome, where the target protein is exposed to the hydrolases present within the lysosome. These enzymes promote the degradation of the target protein into smaller peptides and amino acids, ultimately leading to their complete breakdown within the lysosome. I. Multispecific binding protein form

[0096] As used herein, a "multispecific" binding protein is a binding protein that specifically binds to two or more antigens. A multispecific binding protein that binds to two antigens and / or two different epitopes of different antigens is also referred to herein as a "bispecific" binding protein. A multispecific binding protein that binds to three antigens and / or three different epitopes is also referred to herein as a "trispecific" binding protein. Therefore, multispecific binding proteins can simultaneously bind to two or more different targets, such as membrane-bound CTLA-4 and the target protein. Genetic engineering can be used to design, modify, and generate multispecific binding proteins, or their binding fragments or derivatives, that possess the desired set of binding properties and effector functions.

[0097] In some aspects, the multispecific binding protein of this disclosure comprises a first cell surface binding portion that specifically binds to membrane-bound CTLA-4 on the surface of T cells and a second binding portion that is operatively connected to the first cell surface binding portion and specifically binds to the target protein.

[0098] In some aspects, the first cell surface binding portion of the multispecific binding protein that specifically binds to CTLA-4 on the surface of the T cell is an antibody or an antigen-binding fragment thereof. In some aspects, the first cell surface binding portion of the multispecific binding protein that specifically binds to 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.

[0099] In some respects, the first and second cell surface binding portions of multispecific binding proteins are each independently selected from the group consisting of: antibodies or their antigen-binding fragments (e.g., scFv, Fab, Fab', Fv, F(ab')2), microantibodies, biantibodies, triantibodies, tetraantibodies, tandem di-scFv, tandem tri-scFv, and immunoglobulin single variable domains (ISVs) such as NANOBODY®, V HH (including humanized V) HH ), Camelization V H Single-domain antibodies, domain antibodies, or dAbs.

[0100] In some aspects, the first cell surface binding portion of the multispecific binding protein that specifically binds to CTLA-4 on the surface of the T cell is an antibody or its antigen-binding fragment, an Fc fusion protein, a fragment antigen-binding (Fab) fusion, an immunoglobulin single variable domain (ISV), or a single-chain fragment variable domain (scFv). In some aspects, the first cell surface binding portion of the multispecific binding protein that specifically binds to CTLA-4 on the surface of the T cell is an scFv, wherein the scFv is a linear scFv or a tandem scFv. In some aspects, the first cell surface binding portion of the multispecific binding protein that specifically binds to CTLA-4 on the surface of the T cell is an ISV. In some aspects, the ISV is a V... HH Humanized V HH Or camel-like V H .

[0101] In some aspects, the second binding portion of the multispecific binding protein that specifically binds to the target protein is selected from the group consisting of: antigen, antibody or antigen-binding fragment thereof, autoantibody, Fc fusion protein, Fab, scFv, ISV, AFFIBODY®, or peptide. The second binding portion of the multispecific binding protein that specifically binds to the target protein may be an ISV. In some aspects, the ISV is V. HH Humanized V HH Or camel-like V H .

[0102] ISVs offer several advantages over conventional antibodies: they are approximately ten times smaller than IgG molecules, allowing for the production of correctly folded functional ISVs through in vitro expression in high yields. Furthermore, ISVs are highly stable and resistant to proteases. The characteristics and production of ISVs have been reviewed by Harmsen and De Haard HJ (Applied Microbiology & Biotechnology, November 2007; 77(1):13-22).

[0103] In some respects, the first cell surface binding portion of a multispecific binding protein is an antibody, and the second binding portion of the multispecific binding protein that specifically binds to the target protein is also an antibody. In other respects, the first cell surface binding portion of a multispecific binding protein is an ISV, and the second binding portion of the multispecific binding protein that specifically binds to the target protein is an ISV.

[0104] In some respects, the first cell surface binding portion of a multispecific binding protein is an antibody, and the second binding portion of the multispecific binding protein that specifically binds to the target protein is an ISV. In some respects, the first cell surface binding portion of a multispecific binding protein is an ISV, and the second binding portion of the multispecific binding protein that specifically binds to the target protein is an antibody. In some respects, the first cell surface binding portion of a multispecific binding protein is an antibody fragment, and the second binding portion of the multispecific binding protein that specifically binds to the target protein is an antibody. In other respects, the first cell surface binding portion of a multispecific binding protein is an antibody, and the second binding portion of the multispecific binding protein that specifically binds to the target protein is an antibody fragment. In some respects, the first cell surface binding portion of a multispecific binding protein is an antibody fragment, and the second binding portion of the multispecific binding protein that specifically binds to the target protein is an antibody fragment. In other respects, the first cell surface binding portion of a multispecific binding protein is an antibody fragment, and the second binding portion of the multispecific binding protein that specifically binds to the target protein is an ISV. In other respects, the first cell surface binding portion of multispecific binding proteins is ISV (e.g., V). HH Furthermore, the second binding portion of the multispecific binding protein that specifically binds to the target protein is an ISV (e.g., V). HH ).

[0105] The terms "binding moiety" and "binding domain" are used interchangeably in this document. The binding moiety of a multispecific binding protein can be an antibody, a Fab fragment, an F(ab')2 fragment, an Fv fragment, or an immunoglobulin single variable domain (ISV, such as V). HH ), scFv fragments, fragments containing complementarity-determining regions (CDRs), separated CDRs, or other suitable fragments.

[0106] In some respects, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific binding protein. Antigen-binding fragments of multispecific binding proteins, or their binding fragments or derivatives, can be derived from intact multispecific binding protein molecules using any suitable standard technique such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and (optionally) constant domains of the multispecific binding protein, or its binding fragments or derivatives, using any suitable standard technique such as proteolytic digestion or recombinant genetic engineering techniques. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable conformation or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0107] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid sequence within the variable region of an antibody that confers antigen specificity and binding affinity. Typically, three CDRs (HCDR1, HCDR2, HCDR3) are present in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) are present in each light chain variable region. The term "frame region" or "FR" is known in the art to refer to the non-CDR portion of the variable regions of both the heavy and light chains. Typically, four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) are present in each heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) are present in each light chain variable region.

[0108] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of many well-known schemes, including Kabat et al. (1991), “Sequences of Proteins of Immunological Interest”, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography”, J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, Jan 2003; 27(1):55-77 (“IMGT” numbering scheme); and Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” JMol Biol, 8 Jun 2001; 309(3):657-70 (AHo numbering scheme).

[0109] The boundaries of a given CDR or FR can vary depending on the protocol used for identification. For example, the Kabat protocol is based on structure alignment, while the Chothia protocol is based on structural information. Both the Kabat and Chothia protocols number antibodies based on the length of the most common antibody region sequence, using insertions (e.g., "30a"), and some antibodies show deletions. These two protocols place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The contact protocol is based on the analysis of complex crystal structures and is similar to the Chothia numbering protocol in many ways.

[0110] A “CDR” or “complementation-determining region” or individually designated CDR (e.g., “HCDR1”, “HCDR2”, “HCDR3”) for a given antibody or its region (such as its variable region) should be understood to encompass a complementation-determining region as defined (or specific) by any known protocol. Similarly, a “FR” or “frame region” or individually designated FR (e.g., “FR-H1”, “FR-H2”) for a given antibody or its region (such as its variable region) should be understood to encompass a frame region as defined (or specific) by any known protocol. In some cases, a protocol for identifying a particular CDR or FR is specified, such as a CDR defined by IMGT, Kabat, Chothia, AbM, or contact methods. In other cases, a specific amino acid sequence of the CDR or FR is given. Unless otherwise specified, all specific CDR amino acid sequences mentioned in this disclosure are IMGT CDRs. However, this disclosure also covers alternative CDRs defined by other schemes, such as those determined by abysis Key Annotation (website: abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0111] Unless otherwise specified, the term “antibody” as used herein should be understood to encompass antibody molecules containing two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., complete antibody molecules) and their antigen-binding fragments. Other engineered molecules such as domain-specific binding proteins, single-domain binding proteins, domain-deficient binding proteins, chimeric binding proteins, CDR transplantation binding proteins, biantibodies, triantibodies, tetraantibodies, microantibodies, immunoglobulin single variable domain (ISV) (e.g., monovalent ISV, bivalent ISV, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also included in the term “antigen-binding fragment” as used herein. The term “multispecific antibody” indicates that its binding fragment or derivative binds the antigen-binding sites of two or more antibodies in a single molecule. As used herein, the terms “antigen-binding moiety,” “antigen-binding fragment,” “binding protein,” or “binding moiety,” etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to at least one target antigen to form a complex. In some respects, the binding moiety can refer to one or more fragments of a multispecific binding protein that retain the ability to bind to the membrane-bound CTLA-4 and / or a second target protein or a target protein-specific binding on the surface of T cells.

[0112] In some respects, the antigen-binding fragments disclosed herein are immunoglobulin single variable domains (ISVs), such as domain antibodies, "dAb", and V. HH (including humanized V) HH ), Camelization V H Any suitable fragment of, or any other single variable domain, or any of these. In particular, the antigen-binding fragment of this disclosure may be V. HH Or a fragment thereof.

[0113] The term "immunoglobulin single variable domain" (ISV or ISVD) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which an antigen-binding site is located on and formed by a single immunoglobulin domain. This distinguishes the immunoglobulin single variable domain from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V... H ) and light chain variable structural domain (V L The interaction forms an antigen-binding site. In this case, V H and V L The complementary determinant region (CDR) will facilitate the formation of the antigen binding site; that is, a total of 6 CDRs will participate in the formation of the antigen binding site.

[0114] The term "V" can be used in this article. H "Class 3" ISVs (i.e., those with V) H Three classes of human ancestral sequences (such as DP-47, DP-51, or DP-29) have high sequence homology to ISVs. Furthermore, any type of ISV targeting membrane-bound CTLA-4 and / or target proteins on the surface of T cells, including, for example, those belonging to the so-called "V" group, is also considered. H "Class 4" ISVs (i.e., those with V) H Four types of ethnologous sequences (such as DP-78) have highly sequence-homological ISVs, as described, for example, in WO 2007 / 118 670 A1.

[0115] In particular, ISVs (especially V) HH Sequence and partially humanized V HH The characteristic may be the presence of one or more "hallmark residues" (as described in Table 1 and the subsequent paragraphs describing the single variable domain of NANOBODY® immunoglobulins), making the ISV NANOBODY. ® ISV.

[0116] Therefore, typically, NANOBODY ® ISV (especially V) HH Including (partial or complete) humanized V HH and camel-like V H ) can be defined as an amino acid sequence with a (general) structure: FR1 – CDR1 - FR2 - CDR2 - FR3 - CDR3 -FR4, where FR1 to FR4 refer to framework regions 1 to 4, and where CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, and one or more of the aforementioned marker residues are further defined as shown in Table 1. Specifically, NANOBODY ® ISV (especially V) HH Including (partial) humanized V HH and camel-like V HThe frame sequence (IRS) can be an amino acid sequence with the (general) structure: FR1 – CDR1 -FR2 - CDR2 - FR3 - CDR3 - FR4, where FR1 to FR4 refer to frame regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, as further defined herein. More specifically, the ISV can be an amino acid sequence with the following (general) structure: FR1 – CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, where FR1 to FR4 refer to frame regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3.

[0117] ISVs can specifically bind to and / or target CTLA-4 and / or target proteins on the surface of T cells (as defined herein) and / or target CTLA-4 and / or target proteins. Suitable fragments of the ISV and peptides may also be used, comprising or substantially consisting of one or more such ISVs and / or suitable ISV fragments. The term “immunoglobulin single variable domain (ISV)” encompasses NANOBODY® V as described in WO 08 / 020079 or WO 09 / 138519. HH And therefore, in one respect, V HH Humanized V HH Or camel-like V H (e.g., Camel-turned-human V) H ) or typically sequence-optimized V HH (For example, optimizations for chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression).

[0118] Typically, NANOBODY® immunoglobulin single variable domain (ISV) (especially V) HH Sequence, including (partially) humanized V HH Sequence and Camelization V H A sequence characterized by the presence of one or more “signature residues” (as described herein) in one or more frame sequences (also as further described herein). Therefore, generally, a NANOBODY® ISV can be defined as an immunoglobulin sequence having the following (general) structure. FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to framework regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, with one or more marker residues as further defined herein.

[0119] Specifically, NANOBODY® ISV can be an immunoglobulin sequence having the following (universal) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to frame regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively. The frame sequence is further defined in this paper.

[0120] More specifically, NANOBODY® ISV can be an immunoglobulin sequence having the following (universal) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to frame regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. According to the Kabat number, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 are selected from the marker residues mentioned in Table 1 below. Table 1: Key residues in Nanobody® ISV.

[0121] In some respects, the first and second binding portions of a multispecific binding protein each independently contain an antigen-binding fragment, said antigen-binding fragment comprising at least one variable domain of an antibody or an antigen-binding fragment covalently linked to at least one constant domain. The variable domain can have any size or amino acid composition and will typically contain at least one CDR adjacent to or in frame with one or more frame sequences. L V of domain association H In the antigen-binding fragment of the domain, V H Domain and V L The domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain V. H - V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment may contain monomer V. H or V L Structural domain.

[0122] Non-limiting exemplary configurations of variable and constant domains that can be found within antigen-binding fragments include: (i) V H -CH1;(ii) V H -CH2;(iii) V H -CH3;(iv) V H -CH1-CH2;(v) V H -CH1-CH2-CH3;(vi) V H -CH2-CH3;(vii) V H -CL;(viii) V L -CH1;(ix) V L -CH2;(X) V L -CH3;(xi) V L -CH1-CH2;(xii) V L -CH1-CH2-CH3;(xiii) V L -CH2-CH3; and (xiv) V L -CL. In any configuration of the variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly connected to each other, or they may be connected via complete or partial hinges or linker regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that create a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of a multispecific binding protein may comprise the domains listed above and / or one or more monomers V. H or V L Homodimers or heterodimers (or other polymers) of any variable and constant domain configurations with non-covalent association of domains (e.g., via disulfide bonds).

[0123] In some respects, the first and second cell surface binding portions of multispecific binding proteins are each independently scFv. The single-chain Fv (“scFv”) polypeptide is covalently linked to V H :V L Heterodimers, which are typically composed of gene fusion compounds (including V-type molecules linked by peptide-encoded linkers), H and V LEncoding gene) expression. Human scFv fragments include CDRs maintained in an appropriate conformation, for example, through the use of gene recombination technology. Bivalent and multivalent multispecific binding proteins, or their binding fragments or derivatives, can be spontaneously formed by association with monovalent scFvs, or can be generated by coupling monovalent scFvs via peptide linkers, such as divalent sc(Fv)2. “dsFv” is a V stable by a disulfide bond. H :V L Heterodimer. “(dsFv)2” represents two dsFvs coupled by a peptide linker.

[0124] In some respects, the first and second cell surface binding portions of a multispecific binding protein are each independently Fab. The term "Fab" refers to a binding protein or its binding fragment with a molecular weight of about 50,000 Da and possessing antigen-binding activity, wherein in a fragment obtained by treating IgG with papain, about half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0125] In some respects, the first and second cell surface binding portions of multispecific binding proteins are each independently Fab or F(ab'')2. The term "F(ab'')2" refers to a binding protein or its binding fragment with an antigen-binding activity of approximately 100,000 Da, which, in the case of fragments obtained by treating IgG with pepsin, is slightly larger than Fab bound via disulfide bonds in the hinge region. The term Fab'' refers to a binding protein or its binding fragment with an antigen-binding activity of approximately 50,000 Da, obtained by cleaving the disulfide bonds in the hinge region of F(ab'')2.

[0126] In some respects, the first and second cell surface binding portions of a multispecific binding protein are each independently an immunoglobulin single variable domain (ISV). Embodiments of the immunoglobulin single variable domain (ISV) include obtaining a self-weight chain antibody (V... HH The variable domain of ) and the variable domain of antibodies derived from naturally occurring antibodies lacking light chains (V HH ISVs can be derived from conventional four-chain antibodies or engineered ISVs. ISVs can originate from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. ISVs can be naturally occurring ISVs, present in heavy chain antibodies lacking the light chain. In particular, camel species (e.g., camels, dromedaries, llamas, alpacas, and guanacos) produce naturally occurring heavy chain antibodies lacking the light chain. Camel heavy chain antibodies also lack the CH1 domain.

[0127] In some exemplary aspects, the method of this disclosure includes contacting a T cell with a bispecific ISV construct, wherein the bispecific ISV construct comprises: a) a first ISV that specifically binds to a membrane-bound CTLA-4 on the surface of the T cell; and b) a second ISV that specifically binds to a target protein, such that the bispecific ISV construct binds to both the membrane-bound CTLA-4 and the target protein on the surface of the T cell.

[0128] In some exemplary aspects, the methods disclosed herein include reacting T cells with a bispecific NANOBODY® ISV (such as V... HH Including humanized V H Or camel-like V H ) contact, wherein the bispecific NANOBODY ® The ISV contains: a) a first NANOBODY® ISV (such as V) that specifically binds to CTLA-4 on the surface of T cells. HH Including humanized V H Or camel-like V H b) and a second NANOBODY® ISV (such as V) that specifically binds to the target protein. HH Including humanized V H Or camel-like V H This makes the bispecific NANOBODY ® ISV binds to the membrane on the surface of T cells, including CTLA-4 and target proteins.

[0129] Techniques for preparing multispecific binding proteins (e.g., multispecific antibodies) include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540; WO 93 / 08829 and Traunecker, A. et al., EMBOJ 10 (1991) 3655-3659) and engineered “knob-in-hole” structures (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by: engineering electrostatic redirection effects to prepare binding protein Fc-heterodimer molecules (WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan, M. et al. Science [Science] 229 (1985) 81-83); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny, SA et al., J. Immunol. [Journal of Immunology] 148 (1992) 1547-1553); using “dual antibody” techniques to prepare multispecific binding protein fragments (see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90 (1993) 6444-6448); and using single-chain Fv (scFv) dimers (see, for example, Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and the preparation of trispecific binding proteins, as described, for example, in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

[0130] Several recombinant multispecific binding protein forms have been developed, for example, by fusing, for example, IgG binding protein forms with single-chain domains (see Kontermann RE, mAbs [monoclonal antibodies] 4:2, (2012) 1-16). The variable domain V is described in WO2009080251 and WO 2009080252. L and V H A multispecific binding protein in which the constant domains CL and CH1 are replaced by each other.

[0131] One approach to avoid mismatch byproduct problems, known as the "mortar and pestle structure," aims to force the pairing of two dissimilar binding protein heavy chains by introducing mutations into the CH3 domain to modify the contact interface. On one chain, a large amino acid is replaced with an amino acid with a short side chain to produce a "mortar." Conversely, an amino acid with a large side chain is introduced into the other CH3 domain to produce a "pestle." High yields of heterodimer formation ("pestle-mortar") and homodimer formation ("mortar-mortar" or "pestle-pestle") have been observed by co-expressing these two heavy chains (and two identical light chains, which must be adapted to both heavy chains) (Ridgway JB, Presta LG, Carter P; and WO 1996027011). The percentage of heterodimers can be further increased by reconstructing the interaction surface of the two CH3 domains using phage display methods and introducing disulfide bridges to stabilize the heterodimers (Merchant AM et al., Nature Biotech 16 (1998) 677-681; Aτwell S, Ridgway JB, Wells JA, Carter P., J Mol Biol 270 (1997) 26-35). For example, a novel method for the mortar-and-stick technique is described in EP 1870459A1. Xie, Z. et al., J Immunol Methods 286 (2005) 95-101 mention a form of multispecific binding protein, using scFv, combined with the mortar-and-stick technique for the Fc moiety.

[0132] In some respects, the CH3 domains of the heavy chains of multispecific binding proteins are altered using a "mortar and pestle" technique, which is described in detail in several embodiments, such as WO 96 / 027011, WO 98 / 050431, Ridgway JB et al., Protein Engineering 9 (1996) 617-621, Merchant AM et al., Nat Biotechnol 16 (1998) 677-681. In this approach, the interaction surfaces of the two CH3 domains are altered to increase heterodimerization of the two heavy chains containing the two CH3 domains. Each of the two CH3 domains can be a "mortar and pestle," and the other a "pestle." The introduction of disulfide bridges can be used to stabilize heterodimers (Merchant A.M et al., Nature Biotech 16 (1998) 677-681, Atwell, S. et al., J.Mol. Biol. 270 (1997) 26-35) and increase yield.

[0133] The Fc domain of a bispecific antibody can be engineered to promote heterodimerization over homodimerization. For example, the heavy chain constant region of the first heavy-light chain pair can contain an amino acid sequence different from that of the heavy chain constant region of the second heavy-light chain pair, wherein the different amino acid sequence is engineered to promote heterodimerization of the heavy chain constant region. Implementations include club-and-socket structural mutations or charge pair mutations. Alternatively, the heavy chain constant region of the first heavy-light chain pair can be identical to that of the second heavy-light chain pair, in which case both homodimers and heterodimers are expected to assemble, subsequently separated during antibody manufacturing using one or more purification steps to isolate the desired heterodimer containing an anti-CTLA-4 arm and an anti-target protein arm.

[0134] Multispecific binding proteins with various isotypes and different constant regions can be provided. The Fc region of the antibody is recognized by the Fc receptor and determines the antibody's ability to mediate cellular effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent phagocytosis (ADCP). These cellular effector functions involve recruiting Fc receptor-carrying cells to target cell sites, resulting in the killing of antibody-bound cells. II. CTLA-4 binding site

[0135] In some aspects, the multispecific binding protein of this disclosure includes a binding domain or portion that binds to CTLA-4 (e.g., human CTLA-4) to promote lysosomal targeting. In some aspects, CTLA-4 is an endogenous cell membrane-binding surface receptor expressed on the surface of T cells. In some aspects, the cell membrane-binding surface receptor is CTLA-4 expressed on the surface membrane of T cells.

[0136] An exemplary CTLA-4 binding moiety may be derived from a CTLA-4 antibody, obtained by immunizing mice with natural CTLA-4 or a full-length recombinant CTLA-4 peptide. Alternatively, CTLA-4 or fragments thereof may be generated and modified using biochemical techniques and used as an immunogen. In some aspects, the immunogen may be a peptide derived from the N-terminus or C-terminus of CTLA-4. In some aspects, the immunogen may be a recombinant CTLA-4 peptide expressed in prokaryotes (e.g., *Escherichia coli*) or in eukaryotic or mammalian cells (e.g., Chinese hamster ovary (CHO) cells). In other aspects, the extracellular domain of human CTLA-4 is used to generate the CTLA-4 antibody. In some aspects, the CTLA-4 antibody may be obtained by immunizing transgenic mice expressing a human immune profile (e.g., VELOCIMMUNE® mice from Regeneron). VELOCIMMUNE® mice contain a genome with variable regions of human heavy and light chains, which are operatively linked to endogenous mouse constant region loci, enabling the mice to produce antibodies containing both human and mouse constant regions in response to antigenic stimulation. The DNA encoding the heavy and light chain variable regions of the CTLA-4 antibody can be isolated and incorporated into the multispecific binding protein of this disclosure.

[0137] In some respects, the CTLA-4 binding moiety comprises a variable domain of CTLA-4 antibodies known in the art. For example, the multispecific binding protein of this disclosure may comprise a CTLA-4 binding moiety containing the amino acid sequence of a known anti-CTLA-4 binding protein (e.g., ipilimumab or tremelinumab). In other respects, the CTLA-4 binding protein is a bioequivalence of a known binding protein. For a bioequivalent CTLA-4 binding protein, the contained amino acid sequence may differ from that of a known CTLA-4 binding protein, but retains the ability to bind CTLA-4. When compared to the parental sequence, such a variant CTLA-4 binding protein contains one or more amino acid additions, deletions, or substitutions, but exhibits biological activity substantially equivalent to that of a known CTLA-4 binding protein. For example, if two CTLA-4 binding proteins are pharmaceutical equivalents or pharmaceutical substitutes, they are considered bioequivalent if, when administered under similar experimental conditions at the same molar dose (as a single or multiple dose), their absorption rate and extent do not show significant differences. If some CTLA-4 binding proteins are equivalent in their extent of absorption but not in their rate of absorption, and can still be considered bioequivalent, they will be considered equivalents or drug substitutes because this difference in absorption rate is intentional and reflected in the labeling, not necessary for achieving effective bodily drug concentrations for long-term use, and medically considered irrelevant to the specific drug product under investigation. Bioequivalent variants of known CTLA-4 antibodies can be constructed, for example, by making various substitutions or deletions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other cases, bioequivalent CTLA-4 binding moieties may include variants containing amino acid changes that modify the glycosylation characteristics of known CTLA-4 binding proteins, such as mutations that eliminate or remove glycosylation.

[0138] In some aspects, the first cell surface binding portion that specifically binds to membrane-bound CTLA-4 on the surface of T cells comprises a variable domain of a CTLA-4 antibody or an antigen-binding fragment thereof. In some aspects, the first cell surface binding portion that specifically binds to membrane-bound CTLA-4 on the surface of T cells comprises a CTLA-4 binding portion of a CTLA-4 ligand. In some aspects, the CTLA-4 ligand is selected from the group consisting of CD80 and CD86. In some aspects, the CTLA-4 ligand is an extracellular domain of CD80 or CD86. In some aspects, the first cell surface binding portion comprises a CD80 fragment crystallizable (Fc) fusion polypeptide or a CD86-Fc fusion polypeptide.

[0139] In some aspects, the CTLA-4 binding moiety comprises a CTLA-4 ligand (e.g., CD80 or CD86) or its CTLA-4 binding moiety. In some aspects, the first cell surface target binding moiety is a fusion of a CTLA-4 ligand and an Fc domain (e.g., a CD80-Fc fusion peptide or a CD86-Fc fusion peptide). In some aspects, the Fc domain can be engineered to pair with or heterodimerize with a second binding moiety binding the target molecule. For example, the multispecific binding protein of this disclosure may comprise a first polypeptide containing a fusion of a CTLA-4 ligand and an Fc domain and a binding specificity for the target protein (e.g., V...). HH The second polypeptide (Fc, Fab, or scFv) is fused to a second Fc domain capable of dimerizing with the first Fc domain. Therefore, multispecific binding proteins can promote the internalization and degradation of target proteins through membrane binding of CTLA-4 via the CTLA-4 ligand and the dimerization of the first and second polypeptides.

[0140] Typically, multispecific binding proteins, as described herein, function by binding to membrane-bound CTLA-4 and target proteins with high affinity or cohesion. CTLA-4 dimerization promotes its endocytosis via clathrin-coated pits, leading to enhanced internalization and lysosomal degradation. In some respects, multispecific binding proteins can bind to membrane-bound CTLA-4 and / or soluble target proteins, K... D Less than about 1 μM, as measured by surface plasmon resonance (e.g., at 25°C or 37°C). In some respects, the multispecific binding protein binds to CTLA-4 and / or its target protein, K. D Less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, or less than about 1 nM, as measured by surface plasmon resonance.

[0141] In some respects, the multispecific binding protein described herein binds to CTLA-4 with a dissociation half-life (t½) greater than about 1.1 minutes, as measured by surface plasmon resonance (e.g., at about 25°C or 37°C). In some respects, the multispecific binding protein binds to CTLA-4 and the soluble target protein with t½ greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1200 minutes, as measured by surface plasmon resonance (at 25°C or 37°C).

[0142] In some respects, the multispecific binding proteins described herein contain modified binding moieties to alter binding affinity compared to conjugates containing wild-type binding moieties. In some respects, the modified binding moieties exhibit enhanced binding affinity compared to conjugates containing wild-type binding moieties. In some respects, the modified binding moieties enhance binding affinity at acidic pH compared to conjugates containing wild-type binding moieties. In some respects, the modified binding moieties enhance binding affinity at alkaline pH compared to conjugates containing wild-type binding moieties. In some respects, the modified binding moieties exhibit reduced binding affinity compared to conjugates containing wild-type binding moieties. In some respects, the modified binding moieties reduce binding affinity at acidic pH compared to conjugates containing wild-type binding moieties. In some respects, the modified binding moieties reduce binding affinity at alkaline pH compared to conjugates containing wild-type binding moieties. In some respects, the first cell surface binding portion of the multispecific binding protein binds to membrane-bound CTLA-4 on the surface of T cells with an affinity ranging from about 100 pM to about 1 µM (e.g., about 100 pM to about 1,000 pM, about 1,000 pM to about 0.01 µM, about 0.01 µM to about 0.1 µM, or about 0.1 µM to about 1.0 µM). In other respects, the second binding portion of the multispecific binding protein binds to the target protein with an affinity ranging from about 100 pM to about 1 µM (e.g., about 100 pM to about 1,000 pM, about 1,000 pM to about 0.01 µM, about 0.01 µM to about 0.1 µM, or about 0.1 µM to about 1.0 µM).

[0143] In some respects, the first cell surface binding portion of the multispecific binding protein binds to membrane-bound CTLA-4 on the surface of T cells with an affinity ranging from about 100 pM to about 1 µM. In other respects, the second binding portion of the multispecific binding protein binds to the target protein with an affinity ranging from about 100 pM to about 1 µM. III. pH-sensitive CTLA-4 binding

[0144] In some aspects, the multispecific binding protein of this disclosure includes a CTLA-4 binding moiety that exhibits pH-sensitive binding to CTLA-4. In some aspects, the pH-sensitive binding moiety promotes dissociation of CTLA-4 from the lysosomal compartment, thereby allowing CTLA-4 and / or the multispecific binding protein to recycle back to the cell surface, where it can bind to additional target proteins for degradation. For example, the CTLA-4 binding moiety may include a Fab domain containing one or more mutations that enhance or weaken binding to CTLA-4 under pH conditions different from neutral pH (e.g., at acidic pH). For example, the CTLA-4 binding moiety of a multispecific antibody may be located at CH1, CL, V of the Fab domain. H or V L The region contains mutations, one or more of which increase the affinity of the Fab domain for its antigens in acidic environments (e.g., in tumor microenvironments with pH of about 7.2, 7.0, 6.8, 6.5, 6.3 or lower). When administered to animals, such mutations can lead to an increase in the serum half-life of the multispecific binding protein.

[0145] In some respects, the sensitivity to CTLA-4 binding may increase at acidic pH, thus demonstrating reduced binding to CTLA-4 at lower pH. In another respect, binding to CTLA-4 decreases at pH reflecting the endosome compartment. In some respects, binding to CTLA-4 decreases at pH 5.5 relative to binding at neutral pH (pH 7.0). This decrease in binding at pH 5.5 may be 50% or more of the CTLA-4 binding observed at neutral pH. Changes in anti-CTLA-4 activity (e.g., binding) described herein (e.g., decrease or increase) may be compared to a reference or wild-type antibody. The reference antibody may be an antibody known in the art, such as ipilimumab or trimemumab. The changes may also be relative changes between two different pH levels of a particular antibody composition described herein. pH-sensitive anti-CTLA-4 antibodies can be identified by testing the interaction between CTLA-4 plated on a test plate and a soluble CTLA-4 antibody within a pH range of 4.5 to 7.0, and by selecting antibodies with increased pH sensitivity such that decreased binding is observed at acidic pH. An embodiment of an anti-CTLA-4 antibody exhibiting decreased binding to CTLA-4 at acidic pH includes replacing a tyrosine residue with a histidine residue in or near one or more CDR1-3 regions of at least one of the variable regions of the light and heavy chains of the antibody. See WO 2020214748A1, which is incorporated herein by reference in its entirety.

[0146] In some aspects, the first cell surface binding portion of the multispecific binding protein exhibits pH-dependent binding to membrane-bound CTLA-4 on the surface of the T cells. In some aspects, the second binding portion of the multispecific binding protein exhibits pH-dependent binding to the target protein. In some aspects, the binding of the multispecific binding protein decreases at acidic pH. In some aspects, the first cell surface binding portion of the multispecific binding protein binds to membrane-bound CTLA-4 on the surface of the T cells with an affinity from about 100 pM to about 1 µM. IV. Species selectivity and species cross-reactivity

[0147] In some aspects, the multispecific binding protein of this disclosure employs a CTLA-4 binding moiety that binds to human CTLA-4 but not to CTLA-4 from other species. Alternatively, the multispecific binding protein employs a CTLA-4 binding moiety that binds to human CTLA-4 as well as CTLA-4 from one or more non-human species. For example, the multispecific binding protein may bind to human CTLA-4 and may bind or not (optionally) to one or more of the following CTLA-4 species: mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee. In some aspects, the multispecific binding protein may bind to human CTLA-4 but not to rat and mouse CTLA-4. In other aspects, the multispecific binding protein binds to human CTLA-4 as well as rat and mouse CTLA-4 with similar binding affinity. V. Fc mutations used to silence or enhance effector function and increase half-life

[0148] In some respects, multispecific binding proteins employ CTLA-4 targets that can selectively deplete or stimulate target proteins, for example, in the tumor microenvironment. In one respect, the anti-CTLA-4 binding moiety has increased or decreased Fc-mediated activity. In some respects, target protein depletion can occur through Fc-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP). In another respect, multispecific binding proteins contain Fc domain variants in which at least one amino acid in one or more constant-region domains has been deleted or otherwise altered to provide desired biochemical characteristics, such as reduced or enhanced effector function compared to intact, unaltered antibodies with substantially the same immunogenicity, the ability to non-covalently dimerize, increased ability to localize to tumor sites, decreased serum half-life, increased serum half-life, enhanced binding affinity at acidic pH, decreased binding affinity at non-acidic pH, enhanced binding affinity at non-acidic pH, and decreased binding affinity at non-acidic pH.

[0149] The Fc-mediated effector function of the CTLA-4 binding moiety can be introduced, enhanced, or silenced by any method known in the art. For example, multispecific binding proteins may include an Fc moiety with enhanced effector function. In some aspects, Fc-enhancing mutations (S298A, E333A, and K334A) can be introduced into the CH region of the Fc domain to increase ADCC activity. Alternatively or alternatively, the Fc moiety can be defucosylated to increase antibody-dependent cytotoxicity (ADCC). For example, Biowa's POTELLIGENT® technology uses FUT8 gene knockout CHO cell lines to generate 100% defucosylated antibodies. FUT8 is the only gene encoding al,6-fucosyltransferase, which catalyzes the transfer of fucose from GDP-fucose to GlcNAc in the al,6-linkage of complex oligosaccharides. Probiogen has developed a CHO that is engineered to produce lower levels of fucosylated glycans on MAb, although not through FUT knockout. Probiogen's system introduces a bacterial enzyme that reverts fucose via a de novo synthesis pathway to a sugar-nucleotide that cannot be metabolized by cells. As an alternative, Seattle Genetics has a proprietary feed system that produces lower levels of fucosylated glycans on MAb generated in CHO (and possibly other) cell lines. Xencor has developed XmAb Fc domain technology designed to improve the immune system's elimination of tumor and other pathological cells. The Fc domain has two amino acid changes resulting in up to 40-fold increased affinity for FcyRIIIa. It also increases affinity for FcyRIIa, with the potential to recruit other effector cells, such as macrophages, which play a role in immunity by phagocytosis and digestion of foreign substances (see WO 2019152423A1).

[0150] In some respects, the Fc region of the anti-CTLA-4 binding portion of the multispecific binding protein of this disclosure may employ any field-recognized Fc variant known to confer effector function and / or improve (e.g., reduce or enhance) FcR binding. The Fc variant may include any of the amino acid substitutions disclosed, for example, in the following PCT disclosures: WO 88 / 07089A1, WO 96 / 14339A1, WO 98 / 05787A1, WO 98 / 23289A1, WO 99 / 51642A1, WO 99 / 58572A1, WO 00 / 09560A2, WO 00 / 32767A1, WO 00 / 42072A2, WO 02 / 44215A2, WO 02 / 060919A2, WO 03 / 074569A2, WO 04 / 016750A2, WO 04 / 029207A2, WO 04 / 035752A2, WO 04 / 063351A2, WO 04 / 074455A2、WO 04 / 099249A2、WO 05 / 040217A2、WO 05 / 070963A1、WO 05 / 077981A2、WO 05 / 092925A2、WO 05 / 123780A2、WO 06 / 019447A1、WO 06 / 047350A2, WO 06 / 085967A2 and WO 21 / 016571A2 or U.S. Patent Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784, each incorporated herein by reference in its entirety. In one aspect, the binding polypeptide may comprise an Fc variant (e.g., H268D or H268E) containing an amino acid substitution at EU position 268. On the other hand, the binding peptide may contain amino acid substitutions at EU position 239 (e.g., S239D or S239E) and / or EU position 332 (e.g., I332D or I332Q).

[0151] In some respects, the Fc region of the anti-CTLA-4 binding portion of the multispecific binding protein of this disclosure may contain one or more mutations to reduce or eliminate effector function (see, for example, Zhou et al. (2020) mAbs [Monoclonal Antibody] 12(1): 1814583, which is incorporated herein by reference in its entirety). In some respects, at least one Fc region contains one or more substitutions at amino acid positions 114, 298, 299 and / or 300 according to Kabat numbering (e.g., NNAS mutant – i.e., containing the S298N / T299A / Y300S mutation, or, for example, the A114N glycosylation mutant, A114N).

[0152] In some respects, the Fc region of the anti-CTLA-4 binding moiety of the multispecific binding protein of this disclosure contains one or more mutations to regulate half-life (see, for example, Dall'Acqua et al. (2006) J Biol Chem [Journal of Biochemistry] 281: 23514-24; Zalevsky et al. (2010) Nat Biotechnol [Nature Biotechnology] 28: 157-9; Hinton et al. (2004) J Biol Chem [Journal of Biochemistry] 279: 6213-6; Hinton et al. (2006) J Immunol [Journal of Immunology] 176: 346-56; Shields et al. (2001) J Biol Chem [Journal of Biochemistry] 276: 6591-604; Petkova et al. (2006) Int Immunol [International Journal of Immunology] 18: 1759-69; Datta-Mannan et al. (2007) Drug Metab Disposs 35: 86-94; Vaccaro et al. (2005) Nat Biotechnol 23: 1283-8; Yeung et al. (2010) CancerRes 70: 3269-77; and Kim et al. (1999) Eur J Immunol 29:2819-25. (e.g., T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and / or H435R).

[0153] In some aspects, the multispecific binding protein of this disclosure may have a modified Fc domain. In some aspects, the multispecific binding protein of this disclosure may have a tyrosine residue (Y) at amino acid position 252, according to EU designation. In some aspects, the conjugate may have an aspartic acid residue (D) or a glutamic acid residue (E) at amino acid position 256, according to EU designation. In some aspects, the multispecific binding protein of this disclosure may have tryptophan (W) or glutamine (Q) at amino acid position 307, according to EU designation. In some aspects, according to EU designation, the conjugate may have a phenylalanine residue (F) or a tyrosine residue (Y) at amino acid position 434, according to EU designation.

[0154] In some respects, the multispecific binding protein of this disclosure may have a modified Fc domain comprising any combination of the following four amino acid residues: tyrosine (Y) at amino acid position 252, aspartic acid (D) or glutamic acid (E) at amino acid position 256, tryptophan (W) or glutamine (Q) at amino acid position 307, and phenylalanine (F) or tyrosine (Y) at amino acid position 434; according to EU designation.

[0155] In some aspects, the multispecific binding protein of this disclosure may comprise a modified Fc domain having a combination of amino acid residues selected from the group consisting of: a) tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434; b) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434; c) tyrosine (Y) at amino acid position 252, glutamic acid (E) at amino acid position 256, glutamine (Q) at amino acid position 307, and tyrosine (Y) at amino acid position 434; d) Tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 434; e) Tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and tyrosine (Y) at amino acid position 434; and f) Tyrosine (Y) at amino acid position 252, aspartic acid (D) at amino acid position 256, tryptophan (W) at amino acid position 307, and phenylalanine (F) at amino acid position 434; according to EU designation.

[0156] In some aspects, the multispecific binding protein of this disclosure may comprise a modified Fc domain comprising a tetraamino acid substitution selected from the group consisting of: M252Y / T256D / T307Q / N434Y, M252Y / T256E / T307W / N434Y, M252Y / T256E / T307Q / N434Y, M252Y / T256D / T307Q / N434F, M252Y / T256D / T307W / N434Y and M252Y / T256D / T307W / N434F; according to EU designation.

[0157] In some respects, multispecific binding proteins contain one or more mutations or glycan modifications to regulate Fc-mediated effector function. In other respects, multispecific binding proteins contain one or more mutations to regulate half-life. VI. Species selectivity and species cross-reactivity

[0158] In some aspects, the multispecific binding protein of this disclosure employs a CTLA-4 binding moiety that binds to human CTLA-4 but not to CTLA-4 from other species. Alternatively, the multispecific binding protein employs a CTLA-4 binding moiety that binds to human CTLA-4 as well as CTLA-4 from one or more non-human species. For example, the multispecific binding protein may bind to human CTLA-4 and may bind or not (optionally) to one or more of the following CTLA-4 species: mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee. In some aspects, the multispecific binding protein may bind to human CTLA-4 but not to rat and mouse CTLA-4. In other aspects, the multispecific binding protein binds to human CTLA-4 as well as rat and mouse CTLA-4 with similar binding affinity. VII. Target protein binding portion

[0159] The multispecific binding protein of this disclosure further includes a binding moiety for binding a target protein. As those skilled in the art will understand, when paired with the first CTLA-4 binding moiety to form the multispecific binding protein of this disclosure, the second binding moiety of the multispecific binding protein can promote the internalization and lysosomal degradation of the target protein bound thereto. By specifically binding to the target protein, the multispecific binding protein enables the target protein to be internalized by T cells. The internalized target protein is then transported to the lysosomal compartment, where it undergoes degradation. This mechanism provides a means of targeting and degrading a variety of target proteins or proteins.

[0160] As used herein, because a “target protein” is a protein with a harmful function, degradation would be therapeutically beneficial. In some respects, a target protein is a pathogenic protein or a peptide that causes disease or disease symptoms. In some respects, a target protein is a membrane target protein. In some respects, a target protein is a membrane-bound target protein co-expressed on T cells along with CTLA-4. In other respects, a target protein is expressed on the surface of non-T cells (e.g., antigen-presenting cells). In other respects, a target protein is a soluble protein. Exemplary target proteins or peptides include proteins or peptides secreted by tumors, inflammatory proteins or peptides; signaling molecules, including cytokines, interleukins, interferons, tumor necrosis factor (TNF), growth factors, hormones, neurotransmitters, lipid mediators, activating factors, extracellular matrix (ECM) proteins, Wnt proteins, members of the transforming growth factor-β (TGF-β) family, Notch ligands; and so on.

[0161] In some respects, 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, activating factors, extracellular matrix (ECM) proteins, Wnt proteins, members of the transforming growth factor-β (TGF-β) family, Notch ligands, and immune checkpoint proteins.

[0162] In some respects, the target protein is an antigen (e.g., an autoantigen). An antigen is a molecule that can elicit an immune response. In some respects, the antigen is an autoantigen or self-antigen produced in the subject's cells. For example, the antigen may be a surface marker expressed on a specific cell type, thereby allowing the multispecific binding protein of this disclosure to selectively target and modulate those cells. By binding T cells via CTLA-4 binding, the antigen-targeting multispecific binding protein of this disclosure can enhance immune responses, promote cell-mediated cytotoxicity, or modulate immune cell function in immunotherapy.

[0163] In other respects, the target protein is an antibody (e.g., an autoantibody) or a fragment thereof. An autoantibody is an antibody that specifically binds to one or more antigens prepared or formed by the subject's own body. Autoantibodies mistakenly recognize and target self-antigens, leading to autoimmune diseases. By binding an autoantibody as a second binding component, a multispecific binding protein, or its binding fragment or derivative, can specifically bind to autoantigens associated with autoimmune disorders. This approach offers potential for targeted therapy by redirecting the immune response to self-reactive cells or molecules involved in the autoimmune process. In some respects, the autoantibody disclosed herein is IgM rheumatoid factor (IgM-RF).

[0164] By targeting specific proteins, the multispecific binding proteins disclosed herein can, for example, interfere with protein-protein interactions, disrupt signal transduction pathways, or block protein-mediated cellular functions. In some respects, the target proteins are membrane proteins. Membrane proteins are a class of proteins located within or associated with the cell membrane, playing roles, for example, in cell signal transduction, transmembrane molecular transport, and maintaining the structural integrity of the cell. In some respects, the target proteins are soluble proteins. Soluble proteins are a class of proteins that readily dissolve in an aqueous environment, maintain stability in the cellular environment, and perform various functions.

[0165] In some respects, the target protein is an immune checkpoint protein. In other respects, the target protein is associated with diseases or disorders involving abnormal protein signaling, such as certain cancers or metabolic disorders. The multispecific binding protein of this disclosure can be programmed to target proteins with high affinity and selectivity, enabling precise modulation of abnormal signaling pathways.

[0166] In some respects, the target protein is a pathogenic protein. Pathogenic proteins are those associated with disease development or progression. By promoting the degradation of the target pathogenic proteins, the multispecific binding proteins of this disclosure can neutralize their activity, inhibit their binding to receptors or other molecules, or promote their clearance from the body. This approach is relevant to, for example, the fields of infectious diseases or chronic infectious diseases (such as herpesvirus infections (HSV, CMV, EBV), HIV-1, and HBV infections). In some respects, the multispecific binding protein can be used to treat chronic viral infections. In some respects, the multispecific binding protein of this disclosure can be engineered to target viral or bacterial proteins involved in the pathogenesis. By blocking or neutralizing pathogenic proteins, the multispecific binding protein can help control the spread of disease and limit its impact on the host. In some respects, the multispecific binding protein may comprise a variable domain of a target agent for the target infectious disease, administered co-administered with said agent, or fused with said agent. In some respects, said agent may be palivizumab (e.g., targeting a fusion (F) glycoprotein).

[0167] In some aspects, the target protein may be a tumor-secreted protein. Tumor cells may release proteins that promote tumor growth (such as growth factors), angiogenesis, immune evasion, or metastasis. Multispecific binding proteins targeting said proteins can interfere with their function, inhibit tumor-promoting activity, or enhance anti-tumor immune responses. This approach offers potential for targeted cancer therapies by specifically neutralizing or modulating tumor-secreted proteins that play a key role in tumorigenesis and progression. In some aspects, the tumor-secreted target protein of this disclosure is vascular endothelial growth factor A (VEGFA). In some aspects, the multispecific binding protein may include a variable domain of an agent targeting a specific tumor, co-administered with said agent, or fused with said agent. Exemplary agents may include pegaptanib, bevacizumab, ranibizumab, brolucizumab, and aflibercept (e.g., targeting VEGFA). In some aspects, the target protein is a tumor-secreted protein.

[0168] In some respects, the target protein may be an inflammatory protein. Inflammatory proteins participate in immune responses and can contribute to chronic inflammation, autoimmune disorders, or tissue damage. Multispecific binding proteins can be programmed to target inflammatory proteins and help regulate inflammatory cascades, suppress excessive immune responses, or modulate immune cell function. By selectively binding to and neutralizing inflammatory proteins, the multispecific binding proteins of this disclosure have the potential to suppress inflammation and restore immune balance in a variety of inflammatory conditions. Embodiments of inflammatory conditions include rheumatoid arthritis, dermatitis, and systemic lupus erythematosus (SLE). In some respects, the multispecific binding protein may comprise a variable domain of an agent that targets the target pro-inflammatory protein, administered co-administered with or fused with said agent. Exemplary agents may include eculizumab or ravulizumab (e.g., targeting complement component C5).

[0169] Interleukins (ILs) are a specific group of signaling molecules involved in immune responses and inflammation. The multispecific binding protein of this disclosure can be engineered to target specific ILs or their receptors, thereby modulating their activity and downstream signaling. This approach can be applied to various immune-related disorders, such as autoimmune diseases, allergies, or inflammatory conditions. By interfering with IL signaling, the multispecific binding protein can modulate immune cell activation, cytokine production, or immune cell transport, thus providing potential pathways for therapeutic interventions. In some aspects, the multispecific binding protein may contain a variable domain of a drug that targets a specific IL, be co-administered with said drug, or be fused with said drug. Exemplary agents may include siltuximab (e.g., targeting IL6), mepolizumab or reslizumab (e.g., targeting IL5), secukinumab or ixekizumab (e.g., targeting IL17A), guselkumab, tildrakizumab or risankizumab (e.g., targeting the p19 subunit of IL23), rilonapeptide (e.g., targeting IL1A and IL1B), canakinumab (e.g., targeting IL1B), and ustekinumab (e.g., targeting the p40 subunit of IL12 and IL23).

[0170] In some respects, the target protein may be a Wnt protein. Wnt proteins are a family of secretory signaling molecules that regulate cell proliferation, differentiation, and tissue development. Dysregulation of Wnt signaling is involved in many diseases, including cancer, developmental disorders, and degenerative diseases. Multispecific binding proteins targeting Wnt proteins can modulate their activity, block aberrant signaling pathways, or interfere with Wnt protein interactions. This approach provides a potential therapeutic strategy for diseases driven by aberrant Wnt signaling. In some respects, the multispecific binding protein may contain a variable domain of an agent that targets the target Wnt protein, be co-administered with the agent, or be fused with the agent. Exemplary agents may include vantictumab (e.g., targeting FZD1 / 2 / 5 / 7 / 8).

[0171] In some respects, the target protein may be a cytokine. In some respects, the cytokine may be a member of transforming growth factor-β (TGF-β). Members of the TGF-β family are a group of multifunctional cytokines involved in various cellular processes, including cell growth, differentiation, immune regulation, and tissue repair. Dysregulation of TGF-β signaling is associated with fibrosis, cancer progression, immune disorders, and other diseases. Multispecific binding proteins designed to target members of the TGF-β family can modulate their signaling pathways, inhibit their effects on immune cells or stromal cells, or interfere with TGF-β ligand-receptor interactions. By modulating TGF-β signaling, multispecific binding proteins have potential therapeutic value for a range of diseases associated with TGF-β dysregulation. In some respects, the TGF-β cytokine of this disclosure is TGF-β1. In other respects, the cytokine may be a member of insulin-like growth factor (IGF). Embodiments of IGF include IGF-1 and IGF-2. Other exemplary cytokines include IgE and IgA. In some respects, multispecific binding proteins may include variable domains of agents that target specific cytokines, be co-administered with, or fused with, the agents. Exemplary agents may include omalizumab (e.g., targeting IgE).

[0172] In some respects, the target protein can be a Notch ligand. Notch ligands are cell surface proteins involved in cell communication and tissue development. Dysregulation of Notch signaling is associated with cancer, cardiovascular disease, and neurodegenerative disorders. The multispecific binding protein disclosed herein can disrupt Notch signaling pathways, block ligand-receptor interactions, or regulate downstream gene expression. This approach provides a potential therapeutic strategy for diseases driven by aberrant Notch signaling, with the aim of restoring normal cellular processes and tissue homeostasis.

[0173] In some aspects of this disclosure, the target protein is expressed on the membrane of T cells (e.g., on the same T cells expressing CTLA-4). In other aspects, the target protein is expressed on activated T cells and / or regulatory T (Treg) cells. In some aspects, the target protein comprises membrane-associated proteins, including immune checkpoint proteins and receptors expressed on the surface of T cells. In some aspects, the target protein is an immune checkpoint protein, and the second binding portion comprises an agonist or antagonist immune checkpoint modulator (e.g., an agonist or antagonist immune checkpoint inhibitor). In some aspects, the second binding portion comprises an agonist or antagonist antibody or antigen-binding fragment thereof targeting a receptor involved in immune regulation. In some aspects, the second binding portion comprises an agonist or antagonist ISV targeting a receptor involved in immune regulation.

[0174] In some respects, the target protein is associated with diseases selected from the group consisting of: cancer, autoimmune diseases, inflammatory disorders, infectious diseases, and neurodegenerative disorders. In some respects, the target protein is associated with cancer. In some respects, the target protein is associated with autoimmune diseases. In some respects, the target protein is associated with inflammatory disorders. VIII. pH-sensitive target protein binding

[0175] In some aspects, the multispecific binding protein of this disclosure includes a target-binding moiety exhibiting pH-dependent binding to a target protein. In some aspects, the pH-sensitive binding moiety facilitates the dissociation of the target protein from the multispecific binding protein within the lysosomal compartment, thereby allowing the target protein to degrade in the lysosomal compartment and / or allowing the multispecific binding protein to recycle back to the cell surface, where it can bind to other target proteins for degradation. For example, the target protein binding moiety may include a Fab domain containing one or more mutations that enhance or weaken binding to the target protein under different pH conditions (e.g., at acidic pH) compared to neutral pH. For example, the target protein binding moiety of a multispecific antibody may be located at CH1, CL, V of the Fab domain. H or V L The region contains mutations, one or more of which reduce the affinity of the Fab domain for its antigens in acidic environments (e.g., in tumor microenvironments with pH of about 7.2, 7.0, 6.8, 6.5, 6.3 or lower). When administered to animals, such mutations can lead to an increase in the serum half-life of multispecific binding proteins.

[0176] In some respects, the sensitivity of target protein binding may increase at acidic pH, thus demonstrating reduced binding to the target protein at lower pH. On one hand, binding to the target protein decreases at pH reflecting the endosome compartment. On the other hand, binding to the target protein decreases at pH 5.5 relative to binding at neutral pH (pH 7.0). This decrease in binding at pH 5.5 can be 50% or more of the target protein binding observed at neutral pH. The changes (e.g., decreases or increases) in anti-target protein binding activity (e.g., binding) described herein can be compared to a reference or wild-type antibody. These changes can also be relative changes between two different pH levels of a particular antibody composition described herein. pH-sensitive anti-target protein antibodies can be identified by testing the interaction between a plate-coated target protein and a soluble target protein antibody in a pH range of 4.5 to 7.0, and by selecting antibodies with increased pH sensitivity such that reduced binding is observed at acidic pH. An embodiment of an anti-target protein antibody whose binding to the target protein is reduced at acidic pH includes replacing a tyrosine residue with a histidine residue in or near one or more CDR1-3 regions of at least one of the variable regions of the light and heavy chains of the antibody. See WO2020214748A1, which is incorporated herein by reference in its entirety.

[0177] In some respects, the second binding portion of multispecific binding proteins exhibits pH-dependent binding to the target protein. In other respects, the binding of multispecific binding proteins decreases at acidic pH conditions. IX. Methods for degrading target molecules

[0178] The multispecific binding protein described herein can be used for lysosomal degradation of target proteins mediated by CTLA-4. In some aspects, the method involves using a multispecific binding protein comprising a first binding portion specific for membrane-bound CTLA-4 on T cells and a second binding portion specific for the target molecule. The binding-triggered complex of the multispecific binding protein and membrane-bound CTLA-4 is internalized into T cells via endocytosis, initiating a series of intracellular events.

[0179] After internalization, the CTLA-4 and target protein complex is transported to the early endosome, where sorting and transport processes occur. In the early endosome, the target protein separates from multispecific binding proteins or their binding fragments or derivatives, allowing the target protein to enter the endosome lumen, while CTLA-4 is recycled back to the cell surface. The target protein is now contained within the endosome.

[0180] The next stage involves the maturation of the endosome into a late endosome and subsequently into an endolymphosomal lysosome. Within this compartment, the target protein encounters an environment of gradually increasing acidity, facilitated by the action of proton pumps. The acidic pH triggers the activation of lysosomal enzymes (such as proteases and nucleases), leading to the breakdown of the target protein into smaller peptides and ultimately its complete degradation.

[0181] Following degradation, the resulting peptides, along with any residual fragments of multispecific binding proteins or their binding fragments or derivatives, undergo further processing within endolysosomes. Some peptides can be presented to major histocompatibility complex (MHC) molecules for antigen presentation, facilitating immune surveillance and response. Simultaneously, CTLA-4 is transported back to the cell surface after its recycling pathway, where it can bind to other multispecific binding proteins or their binding fragments or derivatives, as well as target proteins, to initiate further rounds of internalization and degradation.

[0182] By utilizing the CTLA-4 shuttle mechanism, the method enables the efficient and selective degradation of a variety of target proteins, including pathogenic proteins, tumor-secreted proteins, autoantibodies, inflammatory proteins, interleukins, and signaling molecules. The disclosed method can be used to develop targeted therapies for treating diseases such as autoimmune disorders, cancer, and inflammatory conditions by precisely controlling the degradation of specific molecules.

[0183] By promoting the degradation of target proteins, the multispecific binding proteins of this disclosure are particularly useful for treating, preventing, and / or improving any disease or disorder related to or mediated by the expression, signaling, or activity of target proteins, or any disease or disorder that can be treated by CTLA-4-mediated degradation of target proteins in lysosomes. For example, this disclosure provides methods for treating autoimmune diseases, cancer (tumor growth inhibition), chronic viral infections, and other diseases by administering the multispecific binding proteins described herein to patients in need of such treatment. The multispecific binding proteins of this disclosure can be used to treat, prevent, and / or improve diseases or disorders or conditions such as autoimmune diseases, viral infections, or cancer, and / or can be used to improve at least one symptom associated with such diseases, disorders, or conditions. In the context of the treatments described herein, the multispecific binding proteins can be administered as a monotherapy (i.e., as the sole therapeutic agent) or in combination with one or more other therapeutic agents (the embodiments of which are described elsewhere herein).

[0184] In one aspect, this disclosure provides 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 portion that specifically binds to a membrane-bound CTLA-4 on the surface of the T cell; and b) a second binding portion operatively connected to the first cell surface binding portion and specifically binding to the target protein, wherein the binding of the multispecific binding protein to the membrane-bound CTLA-4 on the surface of the T cell promotes the internalization of the target protein by the T cell.

[0185] In some respects, the target protein is degraded in lysosomes after internalization. In other respects, the multispecific binding protein exhibits increased degradation of the target protein compared to a reference binding peptide.

[0186] In some respects, the multispecific binding proteins or their binding fragments or derivatives described herein may be used to treat subjects with cancer or autoimmune disorders and / or inflammatory disorders.

[0187] In some respects, the multispecific binding protein of this disclosure can be used to treat subjects with chronic viral infections. In some respects, the multispecific binding protein can be used to reduce the titer of target proteins in the host via T cell membrane binding to CTLA-4. In some respects, the multispecific binding protein can be administered in therapeutic doses to patients with autoimmune diseases, cancer, or viral infections. Reference binding peptide

[0188] In some respects, compared to the reference binding peptide, the multispecific binding protein of this disclosure exhibits increased internalization and / or degradation of the target protein. In some respects, the reference binding peptide does not contain a first cell surface binding portion that specifically binds to CTLA-4 on the cell surface, but is otherwise identical to the multispecific binding protein. In some respects, compared to the reference binding peptide, the multispecific binding protein exhibits at least 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increased degradation of the target protein. In some respects, compared to the reference binding peptide, the multispecific binding protein is at least 2, 3, 4, 5, 10, 24, 48, or 72 hours faster. Specificity

[0189] The term "specificity" refers to the ability to specifically bind to a given target antigen (e.g., cell surface CTLA-4) (e.g., to induce an immune response). The multispecific binding protein of the present invention contains two or more binding sites (e.g., 2, 3, 4, 5 or more) that specifically bind to the same or different binding sites. In some aspects, the subject multispecific binding protein is specific for two different (e.g., non-overlapping) target binding sites.

[0190] In some aspects, the multispecific binding protein of this disclosure employs a cell surface CTLA-4 binding portion that binds to human CTLA-4 but not to CTLA-4 from other species. Alternatively, the multispecific binding protein employs a CTLA-4 binding portion that binds to human CTLA-4 as well as CTLA-4 from one or more non-human species. For example, the multispecific binding protein may bind to human CTLA-4 and may bind or not (optionally) to one or more of the following CTLA-4 species: mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee. In some aspects, the multispecific binding protein may bind to human CTLA-4 but not to rat and mouse CTLA-4. In other aspects, the multispecific binding protein binds to human CTLA-4 as well as rat and mouse CTLA-4 with similar binding affinity. about or approximately

[0191] The terms “about” or “approximately” mean about 20% of a given value or range (such as about 10%, about 5%, or about 1% or less). application

[0192] As used herein, “administer” means the act of injecting or otherwise physically delivering a substance present outside the body (e.g., the multispecific binding protein described herein) to a patient via injection, through methods such as, but not limited to, pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When managing or treating a disease or its symptoms, the substance is typically administered after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the substance is typically administered before the onset of the disease or its symptoms and may continue for an extended period to delay or reduce the onset or severity of disease-related symptoms.

[0193] In some respects, the multispecific binding proteins described herein are administered via intravenous, subcutaneous, intramuscular, or intradermal injection. Composition

[0194] As used herein, the term "composition" is intended to cover products containing optionally specified amounts of specified ingredients (e.g., multispecific binding protein compositions provided herein), and any products directly or indirectly produced from combinations of optionally specified amounts of specified ingredients.

[0195] In some respects, the pharmaceutical composition comprises the multispecific binding protein described herein and a pharmaceutically acceptable carrier. Effective dose

[0196] "Effective amount" means the amount of an active pharmaceutical ingredient (e.g., the multispecific binding protein of this disclosure) sufficient to achieve the desired physiological outcome in an individual in need of the agent. Effective amounts can vary between individuals depending on the health and physical condition of the individual being treated, the taxonomy of the individual being treated, the formulation of the composition, the assessment of the individual's medical symptoms, and other relevant factors.

[0197] In some aspects, this disclosure provides a method for treating a subject suffering from a disease associated with a target protein or a soluble target protein, the method comprising administering to the subject a therapeutically effective amount of the multispecific binding protein described herein or a pharmaceutical composition comprising the multispecific binding protein described herein. In some aspects, the method comprises binding the multispecific binding protein to membrane-bound CTLA-4 on the surface of T cells, which promotes the internalization of the target protein by the T cell and the transport of the target protein by the T cell to a lysosome within the T cell, allowing the target protein to be degraded within the lysosome, thereby treating the subject's disease. In some aspects, the disease is selected from the group consisting of: cancer, autoimmune diseases, inflammatory disorders, infectious diseases, and neurodegenerative disorders. Subject or patient

[0198] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject may be a mammal, such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In some respects, as used herein, the term “subject” refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, untamed animals, farm animals, sporting animals, and pets. Therapeutic and pharmaceutical compositions

[0199] As used herein, the term "therapy" means any protocol, method, and / or agent that can be used to prevent, manage, treat, and / or improve a disease or its associated symptoms. In some aspects, the term "therapy" means any protocol, method, and / or agent that can be used to modulate or deplete target proteins circulating in a subject. In some aspects, the terms "therapies" and "therapy" mean biological therapies, supportive therapies, and / or other therapies known to those skilled in the art (e.g., medical personnel) that can be used to prevent, manage, treat, and / or improve a disease or its associated symptoms. In other aspects, the term "therapy" means biological therapies, supportive therapies, and / or other therapies known to those skilled in the art (e.g., medical personnel) that can be used to modulate the immune response in a subject to an inflammatory disease or autoimmune disease or its associated symptoms.

[0200] As used herein, the term "treatment" (including both) refers to a reduction or improvement in the progression, severity, and / or duration of disease or related symptoms resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as the administration of the multispecific binding protein described herein). As used herein, the term "treatment" can also refer to alterations to the disease course of a subject undergoing treatment. Therapeutic effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of one or more symptoms, attenuation of the direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement or mitigation of the disease state, and remission or improvement of prognosis. Polynucleotides

[0201] On the one hand, polynucleotides (i.e. nucleic acid molecules) encoding the multispecific binding protein described herein or variants thereof are provided. The polynucleotide variants used herein share approximately 50%, 75%, 80%, 85%, 90%, 93%, 95%, 98%, 99%, or more identity with the polynucleotide encoding the multispecific binding protein described herein.

[0202] A method for preparing a multispecific binding protein is also provided, the method comprising expressing the polynucleotide. Typically, a polynucleotide encoding the multispecific binding protein disclosed herein or a variant thereof is inserted into an expression vector for introduction into a host cell, which can be used to produce the desired amount of the claimed multispecific binding protein. Thus, in some aspects, this disclosure provides an expression vector comprising the polynucleotide disclosed herein and a host cell comprising the vector and the polynucleotide.

[0203] In some respects, the nucleic acid molecule encodes an amino acid sequence of a first cell surface binding portion that specifically binds to CTLA-4 on the surface of CTLA-4 positive cells (e.g., CTLA-4 expressed on the surface of T cells); and an amino acid sequence of a second binding portion that is operatively linked to the first cell surface binding portion and specifically binds to the target protein.

[0204] In some respects, the isolated nucleic acid molecules encode the multispecific binding proteins disclosed in this paper. Expression vectors and host cells

[0205] For the purposes of this specification and claims, the term "vector" or "expression vector" as used herein means a vector used as a medium according to this disclosure for introducing a polynucleotide sequence encoding a multispecific binding protein polypeptide into a cell and expressing said polynucleotide sequence in the cell. Such vectors include, for example, plasmids, bacteriophages, viruses, and retroviruses. Typically, vectors compatible with this disclosure will include selection markers, appropriate restriction sites to facilitate the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0206] In vitro production allows for scaling up to produce large quantities of the desired peptide. Techniques for culturing mammalian cells under tissue culture conditions include, for example, homogeneous suspension culture (e.g., in an airlift reactor or a continuous stirred reactor), or immobilized or embedded cell culture (e.g., in hollow fibers, microcapsules, agarose beads, or ceramic casks). Solutions of the peptide can be purified by conventional chromatographic methods (e.g., gel filtration, ion exchange chromatography, DEAE-cellulose chromatography, and / or (immuno)affinity chromatography) if necessary and / or required.

[0207] One or more genes encoding multispecific binding proteins can also be expressed in non-mammalian cells such as bacteria, yeast, or plant cells. In this respect, it should be understood that a wide variety of single-celled non-mammalian microorganisms, such as bacteria, can also be transformed; that is, those microorganisms capable of growth in culture or fermentation. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of *Escherichia coli* or *Salmonella*; members of the Bacillaceae family, such as *Bacillus subtilis*; *Pneumococcus*; *Streptococcus*; and *Haemophilus influenzae*. It should also be understood that when expressed in bacteria, the polypeptide can become part of an integrity. Polypeptides can be isolated, purified, and then assembled into functional molecules.

[0208] In addition to prokaryotes, eukaryotic cells can also be used. Saccharomyces cerevisiae or common baker's yeast are the most commonly used, although many other strains are usually available.

[0209] In some respects, the vector comprises a nucleic acid molecule encoding: a) an amino acid sequence of a first cell surface binding portion that specifically binds to CTLA-4 on the surface of CTLA-4 positive cells; and b) an amino acid sequence of a second binding portion that is operatively linked to the first cell surface binding portion and specifically binds to the target protein.

[0210] In some respects, at least two vectors contain nucleic acid molecules encoding: a) an amino acid sequence of a first cell surface binding portion that specifically binds to CTLA-4 on the surface of CTLA-4 positive cells; and b) an amino acid sequence of a second binding portion that is operatively linked to the first cell surface binding portion and specifically binds to the target protein.

[0211] In some aspects, both vectors comprise the multispecific binding protein of this disclosure. In some aspects, the first vector comprises a nucleic acid molecule encoding an amino acid sequence of a first cell surface binding portion that specifically binds to CTLA-4 on the surface of CTLA-4-positive cells. In some aspects, the second vector comprises a second binding portion operatively linked to the first cell surface binding portion and specifically binding to the target protein. In some aspects, both vectors comprise the multispecific binding protein of this disclosure.

[0212] In some aspects, the cell comprises a vector containing a nucleic acid molecule encoding: a) an amino acid sequence of a first cell surface binding portion that specifically binds to CTLA-4 on the surface of a CTLA-4 positive cell; and b) an amino acid sequence of a second binding portion operatively linked to the first cell surface binding portion and specifically binding to a target protein. In some aspects, the cell comprises two vectors containing nucleic acid molecules encoding: a) an amino acid sequence of a first cell surface binding portion that specifically binds to CTLA-4 on the surface of a CTLA-4 positive cell; and b) an amino acid sequence of a second binding portion operatively linked to the first cell surface binding portion and specifically binding to a target protein. In some aspects, the cell comprises at least two vectors containing nucleic acid molecules encoding: a) an amino acid sequence of a first cell surface binding portion that specifically binds to CTLA-4 on the surface of a CTLA-4 positive cell; and b) an amino acid sequence of a second binding portion operatively linked to the first cell surface binding portion and specifically binding to a target protein.

[0213] In some respects, the expression vector comprises a nucleic acid molecule encoding a first cell surface binding portion that binds to a second binding portion of the target protein. In some respects, the host cell contains the expression vector.

[0214] It should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive, as the scope of this disclosure will be limited only by the appended claims.

[0215] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. As used herein, when referring to a particular enumerated numerical value, the term “about” means that the value may differ from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0216] Although any methods and materials similar to or equivalent to those described herein may be used in practice with respect to this disclosure, exemplary methods and materials are described hereafter. All publications mentioned herein are incorporated herein by reference in their entirety. Example

[0217] The following embodiments are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and use the methods and compositions described in this disclosure, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at atmospheres or close to atmospheres. Example 1: Design of anti-TNFα / CTLA-4 bispecific antibody construct

[0218] A bispecific antibody construct was designed that is specific to both the target protein (e.g., TNFα) and the expression of CTLA-4 on the cell surface. The light chain and heavy chain amino acid sequences are shown in Table 2 below. Table 2: Light and heavy chain amino acid sequences of anti-TNFα / CTLA-4 bispecific antibody Example 2: Intracellularization and degradation of TNFα via TNFα / CTLA-4 bispecific antibody

[0219] Example 2 tested the ability of the anti-TNFα / CTLA-4 bispecific antibody designed in Example 1 to promote the internalization and degradation of target proteins (e.g., TNFα) within cells. Figure 1 As shown in the diagram.

[0220] Most of the experiments in Example 2 were performed using wild-type Raji cell lines (Raji-empty cells) or Raji cell lines engineered to stably express human CTLA-4 on their cell surface membranes (Raji-CTLA-4 cells) (see Example 2). Figure 14A CTLA-4-specific MFI translocation in [the context of the study]. To confirm that the results were independent of the Raji cell line, samples from two different human donors (“Donor 1” and “Donor 2”; see [reference needed]). Figure 16A and Figure 16B Experiments were conducted on the activation of PBMCs. method CTLA-4 internalization was determined by anti-CLTA-4 antibody analysis using confocal microscopy.

[0221] Raji cells or Raji cells stably expressing human CTLA-4 were incubated in the dark at 37°C for 1 and 4 hours in cell culture medium with 75 µM of anti-CTLA-4 antibody (ipilimumab) labeled with Alexa Fluor 488. After incubation, the culture medium was removed and the cells were washed with PBS. The cells were then fixed with 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) at room temperature for 1 hour, permeabilized with 0.5% Triton X-100 in PBS, washed three times with PBS, stained with the nuclear dye 4',6-diamidinyl-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, MA), and mounted using AquaPoly / Mount.

[0222] To prevent photobleaching during fixation and staining, all incubations were performed in the dark as previously described (Piepenhagen, Microsc Res Tech. 2010). Permeabilized cells were also incubated with fluorescently labeled antibodies against early endosomal antigen 1 (EEA1) or LAMP-1, respectively.

[0223] All photomicrographs were acquired using a Zeiss LSM880 confocal microscope (Carl Zeiss, White Plains, NY) equipped with a 40X Plan-Apo immersion objective. The Alexa Fluor 488 was excited using a 488-nm line from an argon laser and detected using a 515-565-nm bandpass filter. Other fluorescent labels were excited using different nm laser lines and detected using different nm bandpass filters. One or two fields of view were randomly selected from each sample, and the optical stack was recorded. All images were acquired using the same parameters. CTLA-4 internalization was determined by analysis of anti-TNFα / CTLA-4 bispecific antibodies using confocal microscopy.

[0224] In cell culture medium, Raji-empty cells or Raji-CTLA-4 cells stably expressing human CTLA-4 (e.g., empty cells) are placed in the culture medium. Figure 14A (As shown) was incubated in the dark at 37°C for 1 h, 2 h, or 4 h with the following bispecific antibody complex: 25 nM anti-TNFα / CTLA-4 bispecific antibody, 50 nM biotinylated TNFα, and 100 nM streptavidin conjugated to AlexaFlour 488. After incubation, the culture medium was removed, and the cells were washed with PBS.

[0225] Cells were then fixed with 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) at room temperature for 30 minutes, followed by centrifugation onto slides. Samples were then permeabilized with 0.5% Triton X-100 in PBS, washed three times with PBS, stained, and incubated with primary rabbit anti-early endosome antigen 1 (EEA1, an endosome marker) and mouse anti-LAMP-1 (a lysosomal marker) antibodies. After incubation with the primary antibodies, samples were washed three times with PBS, stained, and incubated with hoeschst (a nuclear marker) and fluorescent secondary goat anti-rabbit and goat anti-mouse antibodies. Following staining, samples were washed and mounted using AquaPoly / Mount. To prevent photobleaching during fixation and staining, all incubations were performed in the dark as previously described (Piepenhagen et al., Use of direct fluorescence labeling and confocal microscopy to determine the biodistribution of two protein therapeutics, Cerezyme and Ceredase. Microsc Res Tech. 2010 July;73(7):694-703).

[0226] All photomicrographs were acquired using a Zeiss LSM880 confocal microscope (Carl Zeiss, White Plains, NY) equipped with a 40X Plan-Apo immersion objective. Alexa Fluor 488 was excited using a 488-nm line from an argon laser and detected using a 515-565-nm bandpass filter. Other fluorescent labels were excited using different nm laser lines and detected using different nm bandpass filters. High-magnification images were captured using a Zeiss Airyscan detector array, acquiring each channel sequentially. One or two fields of view were randomly selected for each sample, and the optical stack was recorded. All images were acquired using the same parameters. Images were analyzed in Cellprofiler (Cimini lab, Broad Institute) using the maximum intensity projection of the optical stack. Data were plotted and statistics calculated using RStudio (version 2022.12.0, Posit software). Live-cell imaging with pH-dependent dyes

[0227] Raji-empty cells or Raji-CTLA-4 cells stably expressing human surface CTLA-4 were seeded at a concentration of 200,000 cells / well on 35 mm thick plates coated with 1 mg / mL poly-d-lysine. 2 Cells were placed in glass-bottomed microwell dishes. Cells were allowed to stand overnight to adhere to the dishes. Prior to imaging, recombinant biotinylated TNFα (50 nM), streptavidin-pHrodo red (100 nM), and anti-TNFα / CTLA-4 bispecific antibody (25 nM) were added to each dish. Hoescht (nuclear dye) was added to the medium at a final dilution of 1:5000, and LysoView 633 dye (lysosomal marker dye, Biotium) was added to the medium of some samples at a final concentration of 1X / 1000X stock solution. The dishes were immediately placed in a stage-top incubator system set to 37°C, 5% CO2, and with increased humidity for live-cell imaging.

[0228] All photomicrographs were acquired using a Zeiss LSM780 confocal microscope (Carl Zeiss, White Plains, NY) equipped with a 20X Plan-Apo air immersion objective. Five fields of view were randomly selected from each culture dish, and the optical stack was acquired every 3 minutes for up to two hours. pHrodo red dye was excited using a 561 nm laser line and detected using a bandpass filter. LysoView 633 was excited using a 633 nm laser line, and Hoescht was excited using a 405 nm laser line. They were detected using appropriate bandpass filters. Images were acquired using the same parameters. Live imaging data were analyzed in Cellprofiler (Cimini lab, Broad Institute) using the maximum intensity projection of the optical stack. Data were plotted and statistics were calculated using RStudio (version 2022.12.0, Posit software). TNFα internalization was measured using flow cytometry.

[0229] Raji-empty cells or Raji-CTLA-4 cells stably expressing human surface CTLA-4 were used at 10 5Cells were plated at a concentration of 100 nM / well in U-bottom 96-well plates. Recombinant biotinylated TNFα to a final concentration of 50 nM, streptavidin-Alexa647 to a final concentration of 100 nM, and anti-TNFα antibody or anti-TNFα / CTLA-4 bispecific antibody to a final concentration of 25 or 100 nM were added sequentially to the 96-well plates. After incubating the cells at 37°C for 4 hours, they were washed twice with cold phosphate-buffered saline (PBS) and then subjected to flow cytometry to evaluate Alexa647 fluorescence. The same method was used for 24-hour PHA-stimulated peripheral blood mononuclear cells (PBMCs) with an anti-TNFα / CTLA-4 bispecific antibody to a final concentration of 25 nM. Flow cytometry data were analyzed using FlowJo. TNFα degradation was measured using Western blotting.

[0230] Raji-CTLA4 cells were used at 10 5 Cells were plated at a concentration of 100 cells / well in U-bottom 96-well plates. Recombinant TNFα at a final concentration of 50 nM and anti-TNFα antibody or anti-TNFα / CTLA-4 bispecific antibody at a final concentration of 25 nM were added sequentially to the 96-well plates. After culturing the cells at 37°C for 2 hours, they were washed twice with culture medium and lysed using radioimmunoprecipitation assay (RIPA) buffer. A small number of cells were then incubated for 2, 9, or 22 hours in the presence of DMSO or 100 nM bafloxacin A. At each time point, the cells were washed twice with cold PBS, followed by washing with RIPA buffer to produce cell lysates. The prepared lysates were analyzed for TNFα or β-actin using Western blotting. result

[0231] As confirmed by confocal microscopy, CTLA-4 was rapidly internalized after cells expressing CTLA-4 were incubated with anti-CTLA-4 antibody for one or four hours (but not with isotype controls). Figure 2A , Figure 2B , Figure 3A and Figure 3B ).

[0232] To evaluate the intracellular and lysosomal transport steps of the target protein TNFα after binding to the anti-TNFα / CTLA-4 bispecific antibody construct described in Example 1 via confocal microscopy, such as... Figure 4 The depiction refers to Raji-empty or Raji-CTLA-4 cells ( Figure 14AIt was incubated with a bispecific antibody complex containing biotinylated human TNFα, streptavidin conjugated to AlexaFluor488 (streptavidin-AF488), and an anti-TNFα / CTLA-4 bispecific antibody.

[0233] After incubating the bispecific antibody complex with the cells for 1 hour, 2 hours, or 4 hours ( Figure 6 The samples were fixed and co-stained with rabbit anti-EEA1 and mouse anti-LAMP1 antibodies, fluorescently labeled goat anti-rabbit and goat anti-mouse secondary antibodies, and nuclear marker (Hoechst) using endosome and lysosomal markers, as shown in the representative maximum intensity projection image. Figure 5 and Figure 7 ).like Figures 5-7 As shown, overlap with LAMP1 and / or EEA1 markers indicates internalization of the bispecific antibody complex as early as 1 hour after incubation. Figure 8 (middle and right columns), continue accumulating 2 and 4 hours after the incubation time point ( Figure 5 , Figure 7 and Figure 8 ). Figure 6 Confirmed to overlap with LAMP1 and / or EEA1 markers, the bispecific antibody complexes began to accumulate at the 1-hour post-incubation time point. Most bispecific antibody complexes co-localized with the lysosomal marker LAMP1 at the 2-hour incubation time point. Figure 9 LAMP1 colocalization decreased at the 4-hour incubation time point, potentially attributable to lysosomal degradation of TNFα.

[0234] Unlike non-pH-sensitive fluorophores or pH-sensitive fluorophores that exhibit bright fluorescence at neutral pH, the fluorescence properties of pHrodo red dye provide a ratiometric sensor for measuring pH changes within internal vesicle compartments (like lysosomes and endosomes). Therefore, by conjugating pHrodo-red to a bispecific antibody ( Figure 10 (Upper part) or conjugated to streptobitin-AF488 ( Figure 10 (Lower part) to decorate Figure 4 The bispecific antibody complex described herein was used to confirm, via a second method, that the bispecific antibody complex was indeed absorbed by Raji-CTLA-4 cells and shuttled into low-pH internal compartments (e.g., lysosomes and endosomes). In fact, Figure 11 The overlap between pHrodo-labeling and lysosomal staining (Lysoview) confirmed that some low-pH internal compartments were lysosomes, supporting the imaging of fixed cells. Figure 12This confirmed that pHrodo labeling intensity increased over time, at least during the first hour of incubation. Whether pHrodo-red was conjugated to the bispecific antibody or to streptavidin-AF488, the mean pHrodo-red intensity increased over time after incubation with the bispecific antibody complex. Figure 12 Furthermore, the results were specific to the Raji-CTLA cell line compared to the control cell line (Raji-empty). Figure 13 This is evidenced by the difference in MFI intensity shown.

[0235] To further support the above microscopic examination data, Raji-empty cells or Raji-CTLA-4 cells were compared with 25 nM or 100 nM... Figure 4 The anti-TNFα / CTLA-4 bispecific antibody complex described herein was incubated together with or with an isotype control, and analyzed by flow cytometry. Figure 14B As shown, intracellular TNFα staining was detected only in Raji-CTLA-4 cells treated with the anti-TNFα / CTLA-4 bispecific antibody complex, as demonstrated by a specific CTLA-4 peak shift that was not observed in controls (see [link to relevant documentation]). Figure 14B (The two sub-images at the bottom).

[0236] To confirm these results biochemically, Western blot analysis was performed. Raji-empty or Raji-CTLA-4 cells were incubated with either an anti-TNFα / CTLA-4 bispecific antibody plus TNFα, or an antibody isotype control plus TNFα. Figure 15 As shown, as early as the 9-hour time point, TNFα was degraded in Raji-CTLA-4 cells incubated with the anti-TNFα / CTLA-4 bispecific antibody but not with the isotype control. Furthermore, TNFα was not degraded when a potent lysosomal inhibitor (bafloxacin) was applied to the samples, demonstrating that TNFα is degraded via the lysosomal degradation pathway (see [link to original text]). Figure 15 (At the 22-hour time point).

[0237] As shown in Figure 16, cell internalization can be reenacted in phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMCs) from two different human donors. PBMCs contain approximately 34.6% CD4+. + T cell populations, such as Figure 16A CD4 in + As shown in the diagram. At 4 and 24 hours after incubation with PBMCs (containing multiple T cell populations) activated with TNFα and anti-TNFα / CTLA-4 bispecific antibody, TNFα internalization was observed. Figure 16B ).

[0238] In summary, the anti-TNFα / CTLA-4 bispecific antibody designed in this paper specifically binds to lymphocytes expressing CTLA-4 and its target protein (i.e., TNFα). Subsequently, the CTLA-4 surface expresses the internalized anti-TNFα / CTLA-4 bispecific antibody that binds to TNFα, and it is transported within the endosome and lysosomal compartments via the lysosomal degradation pathway, where TNFα is ultimately degraded. It is envisioned that the target protein binding arm of the multispecific binding protein described herein could be modified to bind to different target proteins, while utilizing cell surface expression of CTLA-4 to maintain internalization and subsequently using the lysosomal transport pathway to degrade the target protein. Example 3: Design of anti-TNFα / CTLA-4 bispecific nanobody construct

[0239] To test whether other designs of multispecific binding proteins that bind to both TNFα and cell surface-expressed CTLA-4 have the same potential for internalizing and degrading TNFα, a bispecific anti-TNFα / CTLA-4 V protein was constructed. HH molecular.

[0240] The sequences of the constructs are shown in Tables 3 and 4. Table 3: Exemplary V HH Construct sequence Table 4: Exemplary NANOBODY® Fusion Construct Sequences Example 4: Intracellularization and degradation of TNFα via TNFα / CTLA-4 bispecific antibody

[0241] Using the method described in Example 2, the ability of the construct prepared in Example 3 to promote the internalization of TNFα expressed on the surface of CTLA-4 cells and subsequently degrade TNFα via the lysosomal degradation pathway was tested. method TNFα internalization was measured using flow cytometry.

[0242] Raji-empty cells and Raji-CTLA-4 cells were mixed at 10 5 Cells / well concentrations were plated in U-bottom 96-well plates. First, recombinant biotinylated TNFα was added to the 96-well plates at a final concentration of 50 nM, then streptavidin-Alexa647 was added to the 96-well plates at a final concentration of 100 nM, and finally anti-TNFα V was added. HH Anti-CTLA-4 V HH Or anti-TNFα / CTLA-4 bispecific V HHThe construct was added to 96-well plates at a final concentration of 25 nM or 100 nM. After culturing the cells at 37°C for 4 hours, they were washed twice with cold phosphate-buffered saline (PBS) and then subjected to flow cytometry to evaluate Alexa647 fluorescence. TNFα degradation was measured using Western blotting.

[0243] Raji-CTLA4 cells were seeded at a concentration of 10⁵ cells / well in U-bottom 96-well plates. Recombinant TNFα was then added to the 96-well plates at a final concentration of 50 nM, followed by anti-TNFα V. HH Anti-CTLA-4 V HH Or anti-TNFα / CTLA-4 bispecific V HH The construct was added to 96-well plates at a final concentration of 25 nM or 100 nM. After culturing the cells at 37°C for 2 hours, they were washed twice with culture medium and lysed using radioimmunoprecipitation assay (RIPA) buffer. A small number of cells were then incubated for 2, 9, and 24 hours in the presence of DMSO or 100 nM bafloxacin A. At each time point, the cells were washed twice with cold PBS, followed by washing with RIPA buffer to produce cell lysates. The prepared lysates were analyzed for TNFα or β-actin using Western blotting.

[0244] In another experiment, Raji-CTLA-4 cells were used at 10 5 Cells / well were plated at a concentration of 1,000 cells / well in U-bottom 96-well plates. Recombinant TNFα was then added to the 96-well plates at a final concentration of 12.5 or 50 nM, and anti-TNFα V was then added. HH Anti-CTLA-4 V HH Or anti-TNFα / CTLA-4 bispecific V HH The construct was added to 96-well plates at a final concentration of 25 nM or 100 nM. After culturing the cells at 37°C for 24, 48, and 72 hours, the cell culture medium was collected and analyzed using Western blotting. result

[0245] like Figure 17 As shown, in the presence of TNFα and 25, 100, or 400 nM of the anti-TNFα / CTLA-4 bispecific V designed in Example 3, HH After co-incubation with the constructs, TNFα internalization was observed in Raji-CTLA-4 cells, as demonstrated by the MFI peak shift, which was observed in contrast to the control V. HH No observations were made when the construct was incubated together with or with Raji-empty cells. Figure 17The graphical representation of the results is shown in Figure 18 middle. Figure 19 This demonstrates that, compared to Raji-empty cells, anti-TNF / CTLA-4 V is effective in Raji-CTLA-4 cells. HH Constructor or V HH The control construct showed an increased fold increase in TNFα internalization. Figure 19 The results demonstrate that the specificity of both TNFα binding and cell surface expression of CTLA-4 is required for TNFα internalization.

[0246] To confirm that TNFα is degraded via the lysosomal pathway after internalization in cells, Western blot analysis was performed. Figure 20 As shown, as early as 9 hours after incubation, when Raji-CTLA-4 cells were inoculated with TNFα plus anti-TNFα / CTLA-4 V... HH TNFα was degraded when the constructs were incubated together. TNFα was not degraded when bafloxacin was applied to the sample, demonstrating that TNFα is degraded via the lysosomal degradation pathway (see [link]). Figure 20 (At 9 and 24 hour points).

[0247] To demonstrate the effect of adding anti-TNFα / CTLA-4 V to Raji-CTLA-4 cells with TNFα. HH After incubation for 24 hours with either the construct or the control, soluble TNFα decreased from the culture medium. TNFα was then added at two different concentrations (12.5 and 50 nM). Figure 21 As shown, in the presence of anti-TNFα / CTLA-4 V HH In cell culture media containing cells incubated with the construct but not with the control, TNFα was reduced.

[0248] Other multispecific binding protein constructs designed in general, such as the anti-TNFα / CTLA-4 V described in this paper. HH The construct can promote the internalization and degradation of target proteins (e.g., TNFα) by binding to cells expressing CTLA-4 on their surface. After binding and internalization, TNF / CTLA-4 V... HH The construct, along with the target protein, is transported and degraded via the lysosomal pathway. Therefore, if the binding arm can be maintained simply by expressing the CTLA-4 binding arm on the cell surface, various binding protein forms can be used to engineer the CTLA-4 multispecific binding protein described herein. As discussed above, any target protein can be engineered into the multispecific binding protein construct described herein.

Claims

1. A method for degrading a target protein, the method comprising: Contact T cells with a multispecific binding protein, wherein the multispecific binding protein comprises: a) The first cell surface binding portion that specifically binds to CTLA-4 on the surface of the T cells; and b) A second binding portion operably linked to the first cell surface binding portion and specifically binding to the target protein. The binding of the multispecific binding protein to the membrane-bound CTLA-4 on the surface of the T cell promotes the internalization of the target protein by the T cell.

2. The method of claim 1, wherein the target protein is degraded in lysosomes after internalization.

3. The method according to claim 1 or 2, wherein the multispecific binding protein expresses increased degradation of the target protein compared to the reference binding polypeptide.

4. The method according to any one of claims 1-3, wherein the first cell surface binding portion that specifically binds to the membrane-bound CTLA-4 on the surface of the T cell comprises a variable domain of a CTLA-4 antibody or an antigen-binding fragment thereof.

5. The method according to any one of claims 1-4, wherein the first cell surface binding portion that specifically binds to membrane-bound CTLA-4 on the surface of the T cell comprises the CTLA-4 binding portion of the CTLA-4 ligand.

6. The method of claim 5, wherein the CTLA-4 ligand is selected from the group consisting of CD80 and CD86.

7. The method of claim 6, wherein the CTLA-4 ligand is an extracellular domain of CD80 or CD86.

8. The method of claim 1, wherein the first cell surface binding portion comprises a CD80 fragment crystallizable (Fc) fusion polypeptide or a CD86-Fc fusion polypeptide.

9. The method according to any one of claims 1-8, wherein 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, activating factors, extracellular matrix (ECM) proteins, Wnt proteins, members of the transforming growth factor-β (TGF-β) family, Notch ligands, and immune checkpoint proteins.

10. The method according to any one of claims 1-9, wherein the target protein is a tumor-secreting protein.

11. The method according to any one of claims 1-10, wherein the target protein is expressed on the membrane of the T cell.

12. The method of claim 11, wherein the T cell line is an activated T cell or a regulatory T (Treg) cell.

13. The method according to any one of claims 1-12, wherein the target protein is an immune checkpoint protein.

14. The method according to any one of claims 1-13, wherein the target protein is associated with a disease selected from the group consisting of: cancer, autoimmune diseases, inflammatory disorders, infectious diseases, and neurodegenerative disorders.

15. The method according to any one of claims 1-14, wherein the target protein is associated with cancer.

16. The method according to any one of claims 1-14, wherein the target protein is associated with an autoimmune disease.

17. The method according to any one of claims 1-14, wherein the target protein is associated with inflammatory disorders.

18. The method according to any one of claims 1-17, wherein the first cell surface binding portion of the multispecific binding protein that specifically binds to the membrane-bound CTLA-4 on the surface of the T cell is an antibody or its antigen-binding fragment, an Fc fusion protein, a fragment antigen-binding (Fab) fusion, an immunoglobulin single variable domain (ISV), or a single-chain fragment variable domain (scFv).

19. The method of claim 18, wherein the first cell surface binding portion of the multispecific binding protein that specifically binds to CTLA-4 on the surface of the T cell is an antibody or an antigen-binding fragment thereof.

20. The method of claim 18, wherein the first cell surface binding portion of the multispecific binding protein that specifically binds to 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.

21. The method of claim 18, wherein the first cell surface binding portion of the multispecific binding protein that specifically binds to the membrane-bound CTLA-4 on the surface of the T cell is an scFv, wherein the scFv is a linear scFv or a tandem scFv.

22. The method of claim 18, wherein the first cell surface binding portion of the multispecific binding protein that specifically binds to CTLA-4 on the surface of the T cell is an ISV.

23. The method of claim 22, wherein the ISV is V HH Humanized V HH Or camel-like V H .

24. The method according to any one of claims 1-23, wherein the second binding portion of the multispecific binding protein that specifically binds to the target protein is selected from the group consisting of: antigen, antibody or antigen-binding fragment thereof, autoantibody, Fc fusion protein, Fab, scFv, ISV, AFFIBODY® or peptide.

25. The method of claim 24, wherein the second binding portion of the multispecific binding protein that specifically binds to the target protein is an antibody or an antigen-binding fragment thereof.

26. The method of claim 24, wherein the second binding portion of the multispecific binding protein that specifically binds to the target protein is an ISV.

27. The method of claim 26, wherein the ISV is V HH Humanized V HH Or camel-like V H .

28. The method according to any one of claims 1-27, wherein the first cell surface binding portion of the multispecific binding protein expresses a pH-dependent binding to membrane-bound CTLA-4 on the surface of the T cell.

29. The method according to any one of claims 1-28, wherein the second binding portion of the multispecific binding protein expresses pH-dependent binding to the target protein.

30. The method according to any one of claims 1-29, wherein the multispecific binding protein exhibits reduced binding at acidic pH.

31. The method according to any one of claims 1-30, wherein the first cell surface binding portion of the multispecific binding protein binds to membrane-bound CTLA-4 on the surface of the T cell with an affinity from about 100 pM to about 1 µM.

32. The method according to any one of claims 1-31, wherein the second binding portion of the multispecific binding protein binds to the target protein with an affinity from about 100 pM to about 1 µM.

33. The method according to any one of claims 1-32, wherein the multispecific binding protein comprises one or more mutations or glycan modifications to modulate the Fc-mediated effector function.

34. The method according to any one of claims 1-33, wherein the multispecific binding protein comprises one or more mutations to regulate its half-life.

35. A multispecific binding protein comprising: a) The first cell surface binding region that specifically binds to CTLA-4 on the surface of T cells; and b) A second binding portion operably connected to the first cell surface binding portion and specifically binding to the target protein. This allows the multispecific binding protein to bind to the membrane-bound CTLA-4 on the surface of the T cell and to the target protein. The binding of CTLA-4 to the membrane on the surface of the T cell promotes the internalization of the target protein by the T cell.

36. The multispecific binding protein of claim 35, wherein the target protein is degraded in lysosomes after internalization.

37. The multispecific binding protein according to claim 35 or 36, wherein the multispecific binding protein expresses an increased degradation of the target protein compared to a reference binding polypeptide.

38. The multispecific binding protein of claim 35, wherein the first cell surface binding portion that specifically binds to CTLA-4 on the surface of the T cell is a CTLA-4 ligand.

39. The multispecific binding protein of claim 38, wherein the CTLA-4 ligand is selected from the group consisting of CD80 and CD86.

40. The multispecific binding protein according to any one of claims 35-39, wherein 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, activating factors, extracellular matrix (ECM) proteins, Wnt proteins, members of the transforming growth factor-β (TGF-β) family, Notch ligands, and immune checkpoint proteins.

41. The multispecific binding protein according to any one of claims 35-40, wherein the target protein is a tumor-secreting protein.

42. The multispecific binding protein according to any one of claims 35-41, wherein the target protein is expressed on the membrane of a T cell.

43. The multispecific binding protein of claim 42, wherein the T cell line is an activated T cell or a Treg cell.

44. The multispecific binding protein according to any one of claims 35-43, wherein the target protein is an immune checkpoint protein.

45. The multispecific binding protein according to any one of claims 35-44, wherein the target protein is associated with a disease selected from the group consisting of: cancer, autoimmune diseases, inflammatory disorders, infectious diseases, and neurodegenerative disorders.

46. ​​The multispecific binding protein according to any one of claims 35-45, wherein the target protein is associated with cancer.

47. The multispecific binding protein according to any one of claims 35-45, wherein the target protein is associated with an autoimmune disease.

48. The multispecific binding protein according to any one of claims 35-45, wherein the target protein is associated with inflammatory disorders.

49. The multispecific binding protein according to any one of claims 35-48, wherein the first cell surface binding portion of the multispecific binding protein that specifically binds to the membrane-bound CTLA-4 on the surface of the T cell is an antibody or its antigen-binding fragment, an Fc fusion protein, a Fab fusion protein, an ISV, or a scFv.

50. The multispecific binding protein of claim 49, wherein the first cell surface binding portion that specifically binds to CTLA-4 on the surface of the T cell is an antibody or its antigen-binding fragment.

51. The multispecific binding protein of claim 49, wherein the first cell surface binding portion that specifically binds to the 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.

52. The multispecific binding protein of claim 49, wherein the first cell surface binding portion that specifically binds to the membrane-bound CTLA-4 on the surface of the T cell is scFv, wherein the scFv is a linear scFv or a tandem scFv.

53. The multispecific binding protein of claim 49, wherein the first cell surface binding portion of the multispecific binding protein that specifically binds to the membrane-bound CTLA-4 on the surface of the T cell is an ISV.

54. The multispecific binding protein according to claim 53, wherein the ISV is V. HH Humanized V HH Or camel-like V H .

55. The multispecific binding protein according to any one of claims 35-54, wherein the second binding portion that specifically binds to the target protein is selected from the group consisting of: antigen, antibody or antigen-binding fragment thereof, autoantibody, Fc fusion, Fab, scFv, ISV, AFFIBODY® or peptide.

56. The multispecific binding protein of claim 55, wherein the second binding portion that specifically binds to the target protein is an ISV.

57. The multispecific binding protein of claim 56, wherein the ISV is V. HH Humanized V HH Or camel-like V H .

58. The multispecific binding protein according to any one of claims 35-57, wherein the first cell surface binding portion of the multispecific binding protein expresses a pH-dependent binding to membrane-bound CTLA-4 on the surface of the T cell.

59. The multispecific binding protein according to any one of claims 35-58, wherein the second binding portion of the multispecific binding protein expresses pH-dependent binding to the target protein.

60. The multispecific binding protein according to any one of claims 35-59, further comprising a modification that allows the multispecific binding protein to bind in a pH-dependent manner to the membrane-bound CTLA-4 on the surface of the T cell.

61. The multispecific binding protein of claim 60, wherein the modification reduces binding at acidic pH.

62. The multispecific binding protein according to any one of claims 35-62, wherein the first cell surface binding portion of the multispecific binding protein binds to the membrane-bound CTLA-4 on the surface of the T cell with an affinity from about 100 pM to about 1 µM.

63. The multispecific binding protein according to any one of claims 35-63, wherein the second binding portion of the multispecific binding protein binds to the target protein with an affinity from about 100 pM to about 1 µM.

64. The multispecific binding protein according to any one of claims 35-61, further comprising one or more mutations or glycan modifications to modulate the Fc-mediated effector function.

65. The multispecific binding protein according to any one of claims 35-64, further comprising one or more mutations to regulate half-life.

66. A pharmaceutical composition comprising a multispecific binding protein according to any one of claims 35-65 and a pharmaceutically acceptable carrier.

67. An isolated nucleic acid molecule, said isolated nucleic acid molecule encoding a multispecific binding protein according to any one of claims 35-65.

68. An expression vector comprising the nucleic acid molecule according to claim 67.

69. A host cell comprising the expression vector according to claim 68.

70. A method of treating a subject suffering from a disease associated with a target protein or a soluble target protein, the method comprising administering to the subject a therapeutically effective amount of a multispecific binding protein according to any one of claims 35-65 or a pharmaceutical composition according to claim 66.

71. The method according to claim 70, wherein: The binding of the multispecific binding protein to the membrane-bound CTLA-4 on the surface of the T cell promotes the internalization of the target protein by the T cell and the transport of the target protein to the lysosome within the T cell, allowing the target protein to be degraded within the lysosome, thereby treating the disease of the subject.

72. The method according to claim 70 or 71, wherein the disease is selected from the group consisting of: cancer, autoimmune diseases, inflammatory disorders, infectious diseases, and neurodegenerative disorders.

73. The method of claim 72, wherein the disease is cancer.

74. The method of claim 72, wherein the disease is an autoimmune disease.

75. The method of claim 72, wherein the disease is an inflammatory disorder.

76. The method according to any one of claims 70-75, wherein the multispecific binding protein is administered via intravenous, subcutaneous, intramuscular, or intradermal injection.

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