Cleavable peptide linkers, fusion proteins, and methods thereof

Abstract

Peptide linkers sensitive to cleavage by cathepsin proteases are provided in the present disclosure. Further provided are fusion proteins comprising cathepsin protease-sensitive peptide linkers. Methods of cleaving a fusion protein via contact with a cathepsin protease are further provided.

Description

[0001] CLEAVABLE PEPTIDE LINKERS, FUSION PROTEINS, AND METHODS THEREOF

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of the earlier filing date of 63 / 566,623, filed March 18, 2024, which is incorporated by reference in its entirety.

[0004] FIELD

[0005] The present disclosure relates to protease cleavable linkers, fusion proteins, and methods of using the same.

[0006] SEQUENCE LISTING

[0007] The instant application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated by reference in its entirety. Said XML copy, created on March 17, 2025, is named 3459W01WO_SQL and is 19 kilobytes in size.

[0008] BACKGROUND

[0009] Fusion proteins are created by joining at least two polypeptide(s) derived from distinct proteins; fusion proteins can be generated in a variety of ways, such as cloning and transcription or chemical synthesis. Fusion proteins offer various advantages, such as supporting purification of cloned genes (e.g., GST protein, FLAG peptide, 6xHis-tag); reporting expression (e.g., luciferase, GFP); increasing stability, catalytic efficiency, and solubility, such as for drug delivery (e.g., antibody-drug conjugates (ADCs)). Peptide linkers can aid in fusion protein design by providing spacing between domains and supporting protein folding while supporting domain interactions, reinforcing stability, and reducing steric hindrance.

[0010] A variety of peptide linkers and types of peptide linkers are known in the art. Cleavable peptide linkers release functional domains via reduction or proteolytic cleavage. For example, in ADCs, cleavable linkers aid an antibody in accurately delivering and releasing a cytotoxic drug at tumor sites. The success of cleavable linkers depends on their ability to discriminate between blood circulation conditions and target cell conditions efficiently. In ADCs, premature cleavage of the linker in the extracellular matrix is often associated with off-target toxicity. Identification of new peptide sequences that allow highly specific cleavage can reduce premature cleavage of the linker at non-tumor sites, hence increasing the safety profiles of ADCs. Therefore, it would be desirable to have more effective peptide linkers at specific and differential pHs. SUMMARY

[0011] Provided herein are peptide linkers comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19.

[0012] Further provided herein are fusion proteins of the formulas:

[0013] Formula I. R1 - L - R2 wherein:

[0014] R1 is a first peptide, polypeptide, or protein;

[0015] L is a peptide linker of the present disclosure; and

[0016] R2 is a second peptide, polypeptide, or protein.

[0017] Formula II. R2 - L - R1 wherein:

[0018] R1 is a first peptide, polypeptide, or protein;

[0019] L is a peptide linker of the present disclosure; and

[0020] R2 is a second peptide, polypeptide, or protein.

[0021] Further provided herein are methods of cleaving a fusion protein, comprising contacting a cathepsin protease with a fusion protein of the present disclosure.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows cleavage of the indicated peptide linkers (SEQ ID NOs: 1-15) on yeast at pH 4.4 by cathepsin B relative to cleavage of GFLGGVR (SEQ ID NO: 20).

[0024] FIG. 2 shows cleavage of the indicated peptide linkers (SEQ ID NOs: 1-12 and 16-19) on yeast at pH 6.4 by cathepsin B relative to cleavage of GFLGGVR (SEQ ID NO: 20).

[0025] DETAILED DESCRIPTION

[0026] Terms

[0027] As used herein, “amino acids” are organic compounds containing amino and carboxylic acid functional groups and can serve as structural units of proteins. As used herein, a “peptide” is a short, linear, single-chain polymer of 2-50 amino acids linked through amino and carboxylate groups via covalent peptide bonds. As used herein, a “polypeptide” is a linear, single-chain polymer of more than 50 amino acids linked through amino and carboxylate groups via covalent peptide bonds. As used herein, a “protein domain” is a polypeptide chain that is typically selfstabilizing and folds independently from other protein elements. Protein domains are typically distinct functional units in a protein. Proteins can have one or more protein domains, and a protein domain can appear in a variety of different proteins. Typically, protein domains vary from about 50 amino acids to 250 amino acids. As used herein, a “protein” contains one or more polypeptides folded into a secondary or tertiary structure. As used herein, a “conjugated protein” is defined as a protein to which another chemical group (e.g., carbohydrate) is attached by either covalent bonding or other interactions.

[0028] As used herein, a “peptide linker” refers to a peptide linking two molecules or moieties (e.g., two heterologous peptides, polypeptides, or proteins). As used herein, a “fusion protein” is two or more heterologous peptides, polypeptides, or proteins (z.e., peptides, polypeptides, or proteins originating from a different source, such as different genes, proteins or polypeptides, organisms, and the like) that are covalently linked to form a single polypeptide.

[0029] As used herein, an “antibody” is an immunoglobulin molecule comprising two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds. As used herein, “antibody fragments” are stable proteolytic cleavage products of full-length antibodies. Chemical fragmentation utilizes reducing agents to break the disulfide bonds within the hinge region and digestion of the antibody with proteases, including pepsin, papain, and ficin. Examples of antibody fragments include Fab (antigen-binding fragment), comprising variable sections that define the specific target that the antibody can bind, and Fc (constant fragment), comprising only the heavy chain CH2 and CH3 domains of an antibody. In embodiments, a fusion protein of the present disclosure includes an antibody or fragment thereof (e.g., CH2 and / or CH3).

[0030] As used herein, “biologically active” molecules or compounds affect biological processes (e.g., binding of a substrate to the active site of an enzyme to catalyze biochemical reactions). As used herein, “sensitive” means activity is affected or changed by various conditions (e.g., activity is affected by different pHs or pH ranges or temperatures). In embodiments, a protease cleavable peptide linker (e.g., a cathepsin B cleavable peptide linker) is pH-sensitive. In embodiments, a protease cleavable peptide linker (e.g., a cathepsin B cleavable peptide linker) is pH sensitive if its cleavage activity (z.e., ability to be cleaved by a protease, such as cathepsin B) is affected or changed at or by different pHs or pH ranges.

[0031] As used herein, proteolytic “cleavage” is the process of breaking peptide bonds between amino acids in proteins and is carried out via enzymes called “proteases.” “Cathepsin proteases” are enzymes that catalyze the hydrolysis of peptide bonds via cleaving amide linkages in an active site, such as through amino acid side chains, for example, a cysteine thiol (also known as a “cysteine cathepsin protease”). Cathepsin proteases show greater activity (z.e., ability to cleave a peptide linker, such as any one of the peptide linkers of SEQ ID NOS: 1-19) in a low or moderately low pH environment, such as a pH of about 4.0-6.8, such as pH 4-6.8, 4.0- 5.5, 4.4-5.0, 4.5-5.0, 4.2-4.6, 6.2-6.6, 4.2, 4.3, 4.4, 4.5, 4.6, 6.2, 6.3, 6.4, 6.5, or 6.6, for example, the low or moderately low pH environment of lysosomes.

[0032] “Cathepsin B” belongs to the cysteine cathepsin protease family and participates in intracellular proteolysis. In humans, cathepsin B is encoded by the CTSB gene and is primarily localized within subcellular endosomal and lysosomal compartments. Mature cathepsin B is composed of a heavy chain of 25-26 kilodaltons (kDa) and a light chain of 5 kDa, which are linked by a disulfide dimer. The proteolytic activity of cathepsin B facilitates direct degradation of several extracellular matrix proteins, including fibronectin, laminin, tenascin C, and type IV collagen. Cathepsin B is upregulated in certain cancers (e.g., brain, lung, prostate, breast, colorectal). Overexpression of cathepsin B is also correlated with invasive and metastatic cancers.

[0033] “Contacting” refers to placement in direct physical association, including both solid and liquid forms. Contacting can occur in vivo or in vitro. In embodiments, contacting occurs in a cell lysosome. “Lysosomes” are membrane-enclosed organelles that contain enzymes capable of breaking down biological polymers (e.g., proteins, nucleic acids, carbohydrates, and lipids). The lysosome has an acidic lumen (z.e., low to moderately low pH) that is optimal for hydrolytic enzymes, such as cathepsin B.

[0034] COMPOSITIONS

[0035] Provided herein are peptide linkers and fusion proteins. In embodiments, peptide linkers are provided herein. In embodiments, peptide linkers provided herein are sensitive to cleavage by a cathepsin protease. Cathepsin proteases are enzymes that catalyze the hydrolysis of peptide bonds via cleaving amide linkages in an active site, such as through amino acid side chains, for example, a cysteine thiol (also known as a cysteine cathepsin protease). In embodiments, peptide linkers provided herein are cleaved by a cathepsin protease selected from the list consisting of: cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin W, and cathepsin Z. In embodiments, peptide linkers provided herein are cleaved by a cysteine cathepsin protease. In embodiments, the peptide linkers are cleaved by a cysteine cathepsin protease selected from the list consisting of: cathepsin B, cathepsin C, cathepsin F, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin W, and cathepsin Z. In embodiments, peptide linkers provided herein are cleaved by cathepsin B.

[0036] In embodiments, peptide linkers provided herein are cleaved by a cathepsin protease at a neutral pH or below a neutral pH. In embodiments, peptide linkers provided herein are cleaved by a cathepsin protease at a neutral pH (e.g., about or approximately a pH of 7.0). In embodiments, peptide linkers provided herein are cleaved by a cathepsin protease at a pH below neutral (e.g., about or approximately below a pH of 7.0). In embodiments, peptide linkers provided herein are cleaved by a cathepsin protease at a pH of 4.0-7.0. In embodiments, peptide linkers provided herein are cleaved by a cathepsin protease at a pH of 4.2-4.6. In embodiments, peptide linkers provided herein are cleaved by a cathepsin protease at a pH of 6.2-6.6. In embodiments, peptide linkers provided herein are cleaved by a cathepsin protease at a pH of 4.4. In embodiments, peptide linkers provided herein are cleaved by a cathepsin protease at a pH of 6.4.

[0037] In embodiments, peptide linkers provided herein are cleaved by a cysteine cathepsin protease at a neutral pH or below a neutral pH. In embodiments, peptide linkers provided herein are cleaved by a cysteine cathepsin protease at a neutral pH (e.g., about or approximately a pH of 7.0). In embodiments, peptide linkers provided herein are cleaved by a cysteine cathepsin protease at a pH below neutral (e.g., about or approximately below a pH of 7.0). In embodiments, peptide linkers provided herein are cleaved by a cysteine cathepsin protease at a pH of 4.0-7.0. In embodiments, peptide linkers provided herein are cleaved by a cysteine cathepsin protease at a pH of 4.2-4.6. In embodiments, peptide linkers provided herein are cleaved by a cysteine cathepsin protease at a pH of 6.2-6.6. In embodiments, peptide linkers provided herein are cleaved by a cysteine cathepsin protease at a pH of 4.4. In embodiments, peptide linkers provided herein are cleaved by a cysteine cathepsin protease at a pH of 6.4.

[0038] In embodiments, peptide linkers provided herein are cleaved by cathepsin B at a neutral pH or below a neutral pH. In embodiments, peptide linkers provided herein are cleaved by cathepsin B at a neutral pH (e.g., about or approximately a pH of 7.0). In embodiments, peptide linkers provided herein are cleaved by cathepsin B at a pH below neutral (e.g., about or approximately below a pH of 7.0). In embodiments, peptide linkers provided herein are cleaved by cathepsin B at a pH of 4.0-7.0. In embodiments, peptide linkers provided herein are cleaved by cathepsin B at a pH of 4.2-4.6. In embodiments, peptide linkers provided herein are cleaved by cathepsin B at a pH of 6.2-6.6. In embodiments, peptide linkers provided herein are cleaved by cathepsin B at a pH of 4.4. In embodiments, peptide linkers provided herein are cleaved by cathepsin B at a pH of 6.4. In embodiments, peptide linkers provided herein comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19. Particularly, in embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 1. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 2. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 3. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 4. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 5. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 6. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 7. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 8. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 9. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 10. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 11. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 12. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 13. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 14. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 15. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 16. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 17. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 18. In embodiments, peptide linkers provided herein comprise the amino acid sequence in SEQ ID NO: 19.

[0039] In embodiments, peptide linkers provided herein are conjugated to a heterologous peptide, polypeptide, or protein (z.e., a peptide, polypeptide, or protein originating from a different source, such as different genes, proteins or polypeptides, organisms, and the like). In embodiments, peptide linkers provided herein are conjugated to one or more heterologous peptides, polypeptides, or proteins. In embodiments, peptide linkers provided herein are conjugated to one heterologous peptide, polypeptide, or protein. In embodiments, peptide linkers provided herein are conjugated to two heterologous peptides, polypeptides, or proteins. In embodiments, peptide linkers provided herein are conjugated to one or more heterologous peptides, polypeptides, or proteins at the C-terminus or N-terminus. In embodiments, peptide linkers provided herein are conjugated to a heterologous peptide, polypeptide, or protein at the C-terminus. In embodiments, peptide linkers provided herein are conjugated to a heterologous peptide, polypeptide, or protein at the N-terminus. In embodiments, peptide linkers provided herein are conjugated to two heterologous peptides, polypeptides, or proteins at the C-terminus and N-terminus.

[0040] Fusion proteins are provided in the present disclosure. In embodiments, the fusion proteins are of the formulas:

[0041] Formula I. R1 - L - R2 wherein:

[0042] R1 is a first peptide, polypeptide, or protein;

[0043] L is a peptide linker of the present disclosure; and

[0044] R2 is a second peptide, polypeptide, or protein.

[0045] Formula II. R2 - L - R1 wherein:

[0046] R1 is a first peptide, polypeptide, or protein;

[0047] L is a peptide linker of the present disclosure; and

[0048] R2 is a second peptide, polypeptide, or protein.

[0049] In embodiments, R1 and R2 of Formulas I or II are heterologous peptides, polypeptides, or proteins (z.e., peptides, polypeptides, or proteins originating from a different source, such as different genes, proteins or polypeptides, organisms, and the like).

[0050] Various peptides, polypeptides, and proteins are possible for R1 or R2 of the present disclosure. In embodiments, the R1 or R2 is a biologically active amino acid sequence, protein fragment, protein domain, antibody, or antibody fragment (e.g., antigen-binding fragment or constant domain fragment). In embodiments, the R1 or R2 is a biologically active amino acid sequence. In embodiments, the R1 or R2 is a protein fragment. In embodiments, the R1 or R2 is a protein domain. In embodiments, the R1 or R2 is an antibody. In embodiments, the R1 or R2 is an antibody fragment (antigen-binding fragment or constant domain fragment).

[0051] In embodiments, fusion proteins provided herein comprise peptide linkers sensitive to cleavage by a cathepsin protease. Particularly, the peptide linkers of fusion proteins provided herein are sensitive to cleavage by a cathepsin protease at a pH of 4.2-4.6. In embodiments, peptide linkers of fusion proteins provided herein comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. Particularly, in embodiments, the peptide linkers of fusion proteins provided herein are sensitive to cleavage by a cathepsin protease at a pH of 6.2-6.6. In embodiments, peptide linkers of fusion proteins provided herein comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and 16-19.

[0052] In embodiments, fusion proteins provided herein comprise peptide linkers sensitive to cleavage by a cysteine cathepsin protease. Particularly, the peptide linkers of fusion proteins provided herein are sensitive to cleavage by a cysteine cathepsin protease at a pH of 4.2-4.6. In embodiments, peptide linkers of fusion proteins provided herein comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. Particularly, in embodiments, the peptide linkers of fusion proteins provided herein are sensitive to cleavage by a cysteine cathepsin protease at a pH of 6.2-6.6. In embodiments, peptide linkers of fusion proteins provided herein comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and 16-19.

[0053] In embodiments, fusion proteins provided herein comprise peptide linkers sensitive to cleavage by cathepsin B. Particularly, the peptide linkers of fusion proteins provided herein are sensitive to cleavage by cathepsin B at a pH of 4.2-4.6. In embodiments, peptide linkers of fusion proteins provided herein further comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15. Particularly, in embodiments, the peptide linkers of fusion proteins provided herein are sensitive to cleavage by cathepsin B at a pH of 6.2-6.6. In embodiments, peptide linkers of fusion proteins provided herein further comprise an amino acid sequence selected from the groups consisting of SEQ ID NOs: 1-12 and 16-19.

[0054] A person of ordinary skill in the art understands that the fusion protein of the present disclosure can be made in a variety of ways using standard laboratory techniques (e.g., Advanced Methods in Molecular Biology and Biotechnology: A Practical Lab Manual (Khalid Z. Masoodi et al., eds., 2021)). In embodiments, Rl, L, and R2 of Formulas I and II are cloned into a vector for transforming, transfecting, or transducing bacteria, insect cells, or mammalian cells for cellbased or in vivo fusion protein expression. Various vectors for transforming, transfecting, or transducing bacteria, insect cells, or mammalian cells are contemplated for making the fusion proteins of the present disclosure, such as plasmid or viral expression vectors. A person of ordinary skill in the art understands that a variety of regulatory or other elements can be included in a plasmid or viral expression vector. In embodiments, regulatory elements are included, such as a promoter, origin of replication, selection marker, and one or more cloning sites. In embodiments, bacteria, insect cells, or mammalian cells are transformed, transfected, or transduced for cell-based or in vivo fusion protein expression using well-understood techniques in the art (e.g., Advanced Methods in Molecular Biology and Biotechnology: A Practical Lab Manual (Khalid Z. Masoodi et al., eds., 2021)). SEQUENCES

[0055] METHODS

[0056] The present disclosure provides peptide linkers and fusion proteins comprising the peptide linkers disclosed herein. Further, the present disclosure provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease selected from the list consisting of: cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin W, and cathepsin Z, with peptide linkers or fusion proteins of the present disclosure. Further, the present disclosure provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease selected from the list consisting of: cathepsin B, cathepsin C, cathepsin F, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin W, and cathepsin Z, with peptide linkers or fusion proteins of the present disclosure. The present disclosure further provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure.

[0057] Further, the present disclosure provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a neutral pH or below a neutral pH. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a neutral pH (e.g., about or approximately a pH of 7.0). The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH below neutral (e.g., about or approximately below a pH of 7.0). The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH of 4.0-7.0. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH of 4.2-4.6. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH of 6.2-6.6. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH of 4.4. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH of 6.4.

[0058] Further, the present disclosure provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a neutral pH or below a neutral pH. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a neutral pH (e.g., about or approximately a pH of 7.0). The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH below neutral (e.g., about or approximately below a pH of 7.0). The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH of 4.0- 7.0. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH of 4.2-4.6. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH of 6.2-6.6. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH of 4.4. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure at a pH of 6.4.

[0059] Further, the present disclosure provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure at a neutral pH or below a neutral pH. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure at a neutral pH (e.g., about or approximately a pH of 7.0). The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure at a pH below neutral (e.g., about or approximately below a pH of 7.0). The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure at a pH of 4.0-7.0. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure at a pH of 4.2-4.6. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure at a pH of 6.2-6.6. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure at a pH of 4.4. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure at a pH of 6.4.

[0060] The present disclosure further provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19.

[0061] The present disclosure further provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure within a lysosome. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure in vitro or in vivo. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure in vitro. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cathepsin protease with peptide linkers or fusion proteins of the present disclosure in vivo. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure within a lysosome. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure in vitro or in vivo. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present disclosure in vitro. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting a cysteine cathepsin protease with peptide linkers or fusion proteins of the present in vivo. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure within a lysosome. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure in vitro or in vivo. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure in vitro. The present disclosure also provides methods of cleaving peptide linkers or fusion proteins of the present disclosure, comprising contacting cathepsin B with peptide linkers or fusion proteins of the present disclosure in vivo.

[0062] EXAMPLES

[0063] A yeast-displayed peptide library was used to identify peptides not known to be sensitive to cathepsin B cleavage. These peptides can be derived from a combination of small motifs found in SEQ ID NOs: 20 and 21. These peptides include GRLVGFD (SEQ ID NO: 1), GRLVGFG (SEQ ID NO: 2), RMLVGFV (SEQ ID NO: 3), RRLYAFL (SEQ ID NO: 4), VFRLLMF (SEQ ID NO: 5), LVGVLLF (SEQ ID NO: 6), VKLYGLG (SEQ ID NO: 7), EQLYLYA (SEQ ID NO: 8), KLFLMIF (SEQ ID NO: 9), NFVIILF (SEQ ID NO: 10), VRLLSLQ (SEQ ID NO: 11), STLMWNV (SEQ ID NO: 12), TWRVDLY (SEQ ID NO: 13), MSLLIGV (SEQ ID NO: 14), VRFLAAA (SEQ ID NO: 15), ENLYFQG (SEQ ID NO: 16), HGWSFHE (SEQ ID NO: 17), VVMMFLH (SEQ ID NO: 18), or VGALVWL (SEQ ID NO: 19).

[0064] Example 1 shows cleavage of the indicated peptide linkers (SEQ ID NOs: 1-15) on yeast at pH 4.4 (FIGURE 1) by cathepsin B relative to cleavage of GFLGGVR (SEQ ID NO: 20).

[0065] Example 2 shows cleavage of the indicated peptide linkers (SEQ ID NOs: 1-12 and 16- 19) on yeast at pH 6.4 (FIGURE 2) by cathepsin B relative to cleavage of GFLGGVR (SEQ ID NO: 20).

[0066] EQUIVALENTS

[0067] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is, therefore, not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Claims

CLAIMSWhat is claimed:

1. A peptide linker, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19.

2. The peptide linker of claim 1, wherein the peptide linker is sensitive to cleavage by a cathepsin protease.

3. The peptide linker of claim 1 or claim 2, wherein the cathepsin protease is a cysteine cathepsin protease.

4. The peptide linker of any one of claims 1-3, wherein the cysteine cathepsin protease is cathepsin B.

5. The peptide linker of any one of claims 1-4, wherein the peptide linker is conjugated to a heterologous peptide, polypeptide, or protein.

6. The peptide linker of claim 5, wherein the peptide linker is conjugated to more than one heterologous peptide, polypeptide, or protein.

7. A fusion protein of the formula:R1 - L - R2 (Formula I), orR2 - L - R1 (Formula II), wherein:R1 is a first peptide, polypeptide, or protein;L is the peptide linker of any one of claims 1-4; andR2 is a second peptide, polypeptide, or protein.

8. The fusion protein of claim 7, wherein R1 or R2 is a biologically active amino acid sequence, protein fragment, protein domain, antibody, or antibody fragment.

9. The fusion protein of claim 8, wherein the peptide linker is sensitive to cleavage by a cathepsin protease at a pH of 4.4.

10. The fusion protein of claim 9, wherein the amino acid sequence of the peptide linker is selected from the group consisting of SEQ ID NOs: 1-15.

11. The fusion protein of claim 8, wherein the peptide linker is sensitive to cleavage by a cathepsin protease at a pH of 6.4.

12. The fusion protein of claim 11, wherein the amino acid sequence of the peptide linker is selected from the group consisting of SEQ ID NOs: 1-12 and 16-19.

13. A method of cleaving a fusion protein, comprising contacting a cathepsin protease with the fusion protein of any one of claims 7-12.

14. The method of claim 13, wherein the cathepsin protease is a cysteine cathepsin protease.

15. The method of claim 13 or claim 14, wherein the cathepsin protease is a cathepsin B.

16. The method of any one of claims 13-15, wherein the contacting occurs in the lysosome.

17. The method of claim 16, wherein the contacting occurs at a pH of 4.0-7.0.

18. The method of claim 16, wherein the contacting occurs at a pH of 4.2-4.6.

19. The method of claim 16, wherein the contacting occurs at a pH of 6.2-6.6.

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