Activatable antibodies for the treatment of autoimmune and inflammatory diseases

Abstract

The present disclosure provides activatable antibodies and antigen binding fragments which bind an immunomodulatory T cell target for use in the treatment of autoimmune and inflammatory diseases, as well as compositions and kits comprising the activatable antibodies.

Description

[0001] Attorney Docket No: TGY-00125

[0002] ACTIVATABLE ANTIBODIES FOR THE TREATMENT OF AUTOIMMUNE AND INFLAMMATORY DISEASES

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims the benefit of U. S. Provisional Patent Application No. 63 / 745,611, filed January 15, 2025, which is incorporated by reference herein in its entirety.

[0005] BACKGROUND

[0006] Autoimmune and inflammatory diseases represent a significant global health burden, affecting millions of individuals worldwide. Autoimmune diseases such as type 1 diabetes (T1D), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis (AD), multiple sclerosis (MS), primary biliary cholangitis, lupus nephritis, Sjorgen’s syndrome-associated dry eye, and / or autoimmune optic neuritis cause chronic inflammation and tissue damage, driven by pathogenic T cell responses. The demand for effective and safer therapies targeting these diseases is substantial, given the growing patient population and the limitations of existing treatment modalities. Biologic agents, particularly monoclonal antibodies (mAbs), have gained prominence in the treatment of immune-mediated diseases due to their high specificity and potential for mechanistic versatility. Some agents can provide short-term immunosuppressive effects, others longer-term immunoregulatory benefits, making them suitable for modulating immune responses across various conditions. Traditional therapies, including immunosuppressants and biologic agents, often lack the specificity required to target only the diseased tissue, leading to systemic effects, increased toxicity, and compromised safety profiles. Therefore, developing precision therapies capable of localizing and activating within affected tissues remains a critical unmet need.

[0007] SUMMARY

[0008] In some aspects, the disclosure provides an activatable antibody comprising:

[0009] a) an antibody, or antigen binding fragment thereof, that specifically binds an immunomodulatory T cell molecule;

[0010] b) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) to the immunomodulatory T cell molecule;

[0011] c) a cleavable peptide coupled to the masking peptide of (b) and the antibody or antigen binding fragment of (a), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,

[0012] d) one or more linker peptides linking (a) and (c) and / or (b) and (c).

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[0015] In some or any of the foregoing aspects, the immunomodulatory T cell molecule modulates one or more of the following properties:

[0016] a) downregulation and / or inhibition of a CD3 / T Cell Receptor (TCR) complex;

[0017] b) activation of effector T cells (Teffs);

[0018] c) activation of regulatory T cells (Tregs);

[0019] d) T cell trafficking;

[0020] e) immune tolerance; and

[0021] f) a combination of (a)-(e).

[0022] In some or any of the foregoing aspects, the activatable antibody inhibits the immunomodulatory T cell molecule and / or activates Tregsand / or enhances immune suppression, and / or inhibits effector T cells. In some or any of the foregoing aspects, the activatable antibody stimulates the immunomodulatory T cell molecule and / or activates Tregsand / or enhances immune suppression, and / or inhibits effector T cells. In some or any of the foregoing aspects, the activatable antibody inhibits interaction of the immunomodulatory T cell molecule with one or more endogenous ligands to and / or inhibits effector T cells. In some or any of the foregoing aspects, the immunomodulatory T cell molecule is selected from a cytokine receptor, a chemokine receptor, an immune checkpoint, a member of the Tumor Necrosis Factor (TNF) superfamily, a member of the Immunoglobulin Superfamily (IgSF), an integrin, or an adhesion molecule. In some or any of the foregoing aspects, the immunomodulatory T cell molecule is selected from CD3, CD28, ICOS, IL-2Ra, IL-2R, IL-7R, IL-10R, PD-1, PD-L1, CTLA-4, TNFR2, CCR4, CCR8, CXCR3, 0X40, OX40L, GITR, TSLPR, CD137 (4-1BB), CD40L (CD154), LAG-3 (CD223), TIM-3 (HAVCR2), VISTA, TIGIT, GARP, LAP, NRP1, BTLA, CD27, CD39, CD47, CD73, CD276, 2B4 (CD244), αEβ7, CD62L, LFA-1, or ICAM-1. In some or any of the foregoing aspects, the immunomodulatory T cell molecule is expressed on Tregs, effector T cells, or both Tregsand effector T cells.

[0023] In some or any of the foregoing aspects, the autoimmune or inflammatory disease is selected from type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, primary biliary cholangitis, lupus nephritis, Sjorgen’s syndrome- associated dry eye, and autoimmune optic neuritis. In some or any of the foregoing aspects, the autoimmune or inflammatory disease tissue is selected from pancreas, joint, intestine, brain, liver, kidneys, heart and eyes.

[0024] In some or any of the foregoing aspects, the masking peptide comprises an amino acid sequence which binds to one or more CDRs of the antibody or antigen binding fragment. In some or any of the foregoing aspects, the masking peptide is an anti-idiotype antibody or an antigen binding fragment thereof. In some or any of the foregoing aspects, the masking peptide is identified by phage display, in vivo immunization, or in silico using artificial-intelligence (Al)-based tools.

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[0027] In some or any of the foregoing aspects, the cleavable peptide is a substrate for a protease in the disease tissue. In some or any of the foregoing aspects, the cleavable peptide comprises a consensus amino acid sequence recognized by the protease in the disease tissue. In some or any of the foregoing aspects, the cleavable peptide is less than 50 amino acids, less than 30 amino acids, less than 20 amino acids or less than 10 amino acids. In some or any of the foregoing aspects, the cleavable peptide comprises a consensus amino acid sequence set forth in Table 9, Table 11, Table 13, or Table 15, or a variant thereof. In some or any of the foregoing aspects, the cleavable peptide comprises an amino acid sequence set forth in Table 10, Table 12, Table 14, Table 16, Table 17 or Table 18, or a variant thereof.

[0028] In some aspects, the disclosure provides an activatable antibody that specifically binds CD3 epsilon (CD3ε) comprising:

[0029] a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 1-3 or SEQ ID NOs: 7-9;

[0030] b) a heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 4-6 or SEQ ID NOs:10-12;

[0031] c) a masking peptide which binds to and inhibits the binding of the antibody to CD3ε;

[0032] d) a cleavable peptide coupled to the masking peptide and either the VL or VH region of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,

[0033] e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

[0034] In some or any of the foregoing aspects, the activatable antibody comprises:

[0035] a) a VL region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs:

[0036] 1-3; and a VH region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 4-6; or

[0037] b) a VL region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs:

[0038] 7-9; and a VH region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 10-12.

[0039] In some or any of the foregoing aspects, the activatable antibody comprises:

[0040] a) a light chain variable (VL) region comprising the sequence of SEQ ID NO: 13; and b) heavy chain variable (VH) region comprising the sequence of SEQ ID NO: 14.

[0041] In some or any of the foregoing aspects, the activatable antibody comprises a human IgG Fc region which binds Fc receptors.

[0042] In some or any of the foregoing aspects, the masking peptide is selected from an amino acid sequence of SEQ ID NOs: 174-191.

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[0045] In some or any of the foregoing aspects, binding of the activatable antibody to CD3ε induces downregulation and / or inhibition of the TCR complex, and / or effector T cell death, and / or expansion and / or proliferation of Tregs, and / or cytokine production, or a combination thereof.

[0046] In some aspects, the disclosure provides an activatable antibody that specifically binds PD-1, comprising:

[0047] a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 15-17;

[0048] b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 18-20;

[0049] c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD- 1;

[0050] d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,

[0051] e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

[0052] In some aspects, the disclosure provides an activatable antibody that specifically binds PD-1, comprising:

[0053] a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 21-23;

[0054] b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 24-26;

[0055] c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD-1:

[0056] d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue: and, optionally,

[0057] e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

[0058] In some aspects, the disclosure provides an activatable antibody that specifically binds PD-1, comprising:

[0059] a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 27-29;

[0060] b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 30-32;

[0061] c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD-1;

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[0064] d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,

[0065] e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

[0066] In some aspects, the disclosure provides an activatable antibody that specifically binds PD-1, comprising:

[0067] a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 33-35;

[0068] b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 36-38;

[0069] c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD-1;

[0070] d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,

[0071] e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

[0072] In some aspects, the disclosure provides an activatable antibody that specifically binds PD-1, comprising:

[0073] a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 39-41;

[0074] b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 42-44;

[0075] c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD-1;

[0076] d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,

[0077] e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

[0078] In some aspects, the disclosure provides an activatable antibody that specifically binds PD-1, comprising:

[0079] a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 45-47;

[0080] b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 48-50;

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[0083] c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD-1;

[0084] d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,

[0085] e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

[0086] In some aspects, the disclosure provides an activatable antibody that specifically binds PD-1, comprising:

[0087] a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 51-53;

[0088] b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 54-56;

[0089] c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD-1;

[0090] d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue: and, optionally,

[0091] e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

[0092] In some or any of the foregoing aspects, binding of the activatable antibody to PD-1 induces effector T cell death.

[0093] In some aspects, the disclosure provides an activatable antibody that specifically binds TNFR2, comprising:

[0094] a) a light chain variable (VL) region comprising a CDR-L1, CDR-L2, and CDR-L3 and heavy chain variable (VH) region comprising a CDR-H1. CDR-H2, and CDR-H3 amino acid sequence set forth in Table 4A;

[0095] b) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) to TNFR2;

[0096] c) a cleavable peptide coupled to the masking peptide of (b) and the antibody or antigen binding fragment of (a), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,

[0097] d) one or more linker peptides linking (a) or (b) with (c), or (b) with (c).

[0098] In some aspects, the disclosure provides an activatable antibody that specifically binds TNFR2, comprising:

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[0101] a) a light chain variable (VL) region comprising a CDR1, CDR2, and CDR3 amino acid sequence set forth in any one of SEQ ID NOs: 106-134;

[0102] b) a heavy chain variable (VH) region comprising a CDR1, CDR2, and CDR3 amino acid sequence set forth in any one of SEQ ID NOs: 135-171;

[0103] c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to TNFR2;

[0104] d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue: and, optionally,

[0105] e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

[0106] In some aspects, the disclosure provides an activatable antibody that specifically binds TNFR2 comprising:

[0107] a) a polypeptide sequence set forth in any one of SEQ ID NOs: 104-105;

[0108] b) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to TNFR2;

[0109] c) a cleavable peptide coupled to the masking peptide of (b) and the antibody or antigen binding fragment of (a), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue: and, optionally,

[0110] d) one or more linker peptides linking (a) with (c), or (b) with (c).

[0111] In some aspects, the disclosure provides an activatable antibody that specifically binds CD2, comprising:

[0112] a) heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 172, b) a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 173, c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to CD2;

[0113] d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,

[0114] e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

[0115] In some or any of the foregoing aspects, the activatable antibody modulates one or more of the following properties:

[0116] a) downregulation and / or inhibition of a CD3 / T Cell Receptor (TCR) complex;

[0117] b) activation of effector T cells (Teffs);

[0118] c) activation of regulatory T cells (Tregs);

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[0121] d) T cell trafficking;

[0122] e) immune tolerance; and

[0123] f) a combination of (a)-(e).

[0124] In some or any of the foregoing aspects, the activatable antibody inhibits CD3, PD-1, TNFR2 and / or CD2, and / or activates Tregsand / or enhances immune suppression, and / or inhibits effector T cells. In some or any of the foregoing aspects, the activatable antibody stimulates CD3, PD-1, TNFR2 and / or CD2 and / or activates Tregsand / or enhances immune suppression, and / or inhibits effector T cells. In some or any of the foregoing aspects, CD3, PD-1, TNFR2 and / or CD2 is expressed on Tregs, effector T cells, or both Tregsand effector T cells.

[0125] In some or any of the foregoing aspects, the autoimmune or inflammatory disease is selected from type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, primary biliary cholangitis, lupus nephritis, Sjorgen’s syndrome- associated dry eye, and autoimmune optic neuritis. In some or any of the foregoing aspects, the autoimmune or inflammatory disease tissue is selected from pancreas, joint, intestine, brain, liver, kidneys, heart and eyes.

[0126] In some or any of the foregoing aspects, the masking peptide comprises an amino acid sequence which binds to one or more CDRs of the antibody or antigen binding fragment. In some or any of the foregoing aspects, the masking peptide is an anti-idiotype antibody or an antigen binding fragment thereof. In some or any of the foregoing aspects, the masking peptide is identified by phage display, in vivo immunization, or in silico using artificial-intelligence (Al)-based tools.

[0127] In some or any of the foregoing aspects, the cleavable peptide is a substrate for a protease in the disease tissue. In some or any of the foregoing aspects, the cleavable peptide comprises a consensus amino acid sequence recognized by the protease in the disease tissue. In some or any of the foregoing aspects, the cleavable peptide is less than 50 amino acids, less than 30 amino acids, less than 20 amino acids or less than 10 amino acids. In some or any of the foregoing aspects, the cleavable peptide comprises a consensus amino acid sequence set forth in Table 9, Table 11, Table 13, or Table 15, or a variant thereof. In some or any of the foregoing aspects, the cleavable peptide comprises an amino acid sequence set forth in Table 10, Table 12, Table 14, Table 16, Table 17 or Table 18, or a variant thereof.

[0128] In some or any of the foregoing aspects, the activatable antibody comprises a linker peptide linking the VH or VL with the cleavable peptide or linking the masking peptide with the cleavable peptide.

[0129] In some or any of the foregoing aspects, the masking peptide is less than 20 amino acids, less than 15 amino acids, less than 10 amino acids or less than 5 amino acids. In some or any of the foregoing aspects, the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VH domain. In some or any of the foregoing

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[0132] aspects, the masking peptide is linked directly to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VH domain. In some or any of the foregoing aspects, the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked directly to the VH domain. In some or any of the foregoing aspects, the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VL domain. In some or any of the foregoing aspects, the masking peptide is linked directly to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VL domain. In some or any of the foregoing aspects, the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked directly to the VL domain.

[0133] In some or any of the foregoing aspects, the linker peptide is comprised of less than 30 amino acids, less than 20 amino acids, or less than 10 amino acids. In some or any of the foregoing aspects, the linker peptide is a GlySer linker. In some or any of the foregoing aspects, the linker peptide is comprised of an amino acid sequence of SEQ ID NOs: 436-438. In some or any of the foregoing aspects, wherein the linker peptide is comprised of about 1 to 30 amino acids.

[0134] In some or any of the foregoing aspects, the antibody is a humanized antibody, a chimeric antibody, or a human antibody. In some aspects, a nucleic acid encodes the activatable antibody of some or any of the foregoing aspects. In some or any of the foregoing aspects, a vector comprises the nucleic acid. In some aspects, the disclosure provides a host cell comprising some or any of the foregoing aspects.

[0135] In some aspects, the disclosure provides a method of producing an activatable antibody, comprising culturing the host cell of some or any of the foregoing aspects under conditions that produce the activatable antibody.

[0136] In some aspects, the disclosure provides a pharmaceutical composition comprising the activatable antibody of some or any of the foregoing aspects and a pharmaceutically acceptable carrier.

[0137] In some aspects, the disclosure provides a kit comprising the activatable antibody of some or any of the foregoing aspects and instructions for use.

[0138] In some aspects, the disclosure provides a method of treating an autoimmune or inflammatory disease in a subject, comprising administering to the subject an activatable antibody or pharmaceutical composition of some or any of the foregoing aspects.

[0139] In some or any of the foregoing aspects, the activatable antibody or pharmaceutical composition is administered to the subject intravenously, intramuscularly, systemically, or locally.

[0140] In some aspects, the disclosure provides use of the activatable antibody or pharmaceutical composition of some or any of the foregoing aspects in the manufacture of a medicament for the treatment of an autoimmune or inflammatory disease.

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[0143] In some aspects, the disclosure provides use of the activatable antibody or pharmaceutical composition of some or any of the foregoing aspects for the treatment of an autoimmune or inflammatory disease in a subject.

[0144] In some aspects, the disclosure provides an activatable antibody comprising:

[0145] a) an antibody, or antigen binding fragment thereof, that specifically binds an immunomodulatory T cell molecule;

[0146] b) at least one masking peptide which binds to and inhibi ts the binding of the antibody or antigen binding fragment of (a) to the immunomodulatory T cell molecule; c) a cleavable peptide coupled to the masking peptide of (b) and the antibody or antigen binding fragment of (a), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally, d) one or more linker peptides linking (a) and (c) and / or (b) and (c).

[0147] In some embodiments, the immunomodulatory T cell molecule is selected from a cytokine receptor, a chemokine receptor, an immune checkpoint, a member of the Tumor Necrosis Factor (TNF) superfamily, a member of the Immunoglobulin Superfamily (IgSF), an integrin, or an adhesion molecule. In some embodiments, the immunomodulatory T cell molecule is selected from CD3, CD28, ICOS, IL-2, IL-2Ra, IL-2R, IL-7R, IL-10R, PD-1, PD-L1, CTLA-4, TNFR2, CCR4, CCR8, CXCR3, 0X40, OX40L, GITR, TSLPR, CD137 (4-1BB), CD40L (CD154), LAG-3 (CD223), TIM-3 (HAVCR2), VISTA, TIGIT, GARP, LAP, NRP1, BTLA, CD27, CD39, CD47, CD73, CD276, 2B4 (CD244), αEβ7, CD62L, LFA-1, ICAM-1, or a combination thereof. In some embodiments, the at least one masking peptide is set forth in SEQ ID NO: 447. In some embodiments, the first masking peptide is linked to the VH domain and the second masking peptide is linked to the VL domain. In some embodiments, the cleavable peptide comprises an amino acid sequence set forth in Table 10, Table 12, Table 14, Table 16, Table 17 or Table 18, or a variant thereof.

[0148] In some aspects, the disclosure provides an activatable antibody that specifically binds human CD3 epsilon (CD3ε) comprising:

[0149] a) a light chain variable (VL) region;

[0150] b) a heavy chain variable (VH) region;

[0151] c) at least one masking peptide which binds to and inhibits the binding of the antibody to CD3ε; d) a cleavable peptide coupled to the at least one masking peptide and either the VL or VH region of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally.

[0152] e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

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[0155] In some embodiments, the VL comprises a CDR-L1 set forth in SEQ ID NO: 1, a CDR-L2 set forth in SEQ ID NO: 2, a CDR-L3 set forth in SEQ ID NO: 4, and wherein the VH comprises a CDR-H1 set forth in SEQ ID NO: 4, a CDR-H2 set forth in SEQ ID NO: 5, and a CDR-H3 set forth in SEQ ID NO: 6. In some embodiments, the VL comprises a CDR-L1 set forth in SEQ ID NO: 531, a CDR-L2 set forth in SEQ ID NO: 532, a CDR-L3 set forth in SEQ ID NO: 533, and wherein the VH comprises a CDR-H1 set forth in SEQ ID NO: 534, a CDR-H2 set forth in SEQ ID NO: 535, and a CDR-H3 set forth in SEQ ID NO: 536. In some embodiments, the at least one masking peptide is set forth in SEQ ID NO: 447 or 449.

[0156] In some embodiments, the cleavable peptide is a substrate for a protease enriched in the pancreas of a subject. In some embodiments, the protease is trypsin. In some embodiments, the cleavable peptide is selected from SEQ ID NO: 448, 234-298, or 549-596.

[0157] In some embodiments, the activatable antibody reduces production of IFNy, TNFa, and IL-6 in the blood compared to a control antibody lacking the at least one masking peptide. In some embodiments, the activatable antibody does not downregulate expression of the CD3 / T cell Receptor (TCR) complex on T cells in the absence of the protease. In some embodiments, the activatable antibody downregulates expression of the CD3 / T cell Receptor (TCR) complex on T cells in the presence of the protease. In some embodiments, the protease is trypsin. In some embodiments, the T cells are CD4+ and / or CD8+ T cells. In some embodiments, the activatable antibody does not induce T cell proliferation in the absence of the protease. In some embodiments, the activatable antibody induces T cell proliferation in the presence of the protease. In some embodiments, the T cells are CD4+ and / or CD8+ T cells. In some embodiments, the T cells are human primary T cells.

[0158] In some embodiments, the at least one masking peptide inhibits binding of anti-CD3 to CD3. In some embodiments, the at least one masking peptide is linked to the cleavable peptide and wherein the cleavable peptide is linked directly to the VL domain. In some embodiments, the at least one masking peptide is linked to the cleavable peptide and wherein the cleavable peptide is linked directly to the VH domain. In some embodiments, the at least one masking peptide comprises a first masking peptide and a second masking peptide. In some embodiments, the first masking peptide is linked to the VH domain and the second masking peptide is linked to the VL domain.

[0159] In some embodiments, the at least one masking peptide comprises an amino acid sequence which binds to one or more CDRs of the antibody or antigen binding fragment of (a). In some embodiments, the masking peptide is an anti-idiotype antibody or an antigen binding fragment thereof. In some embodiments, the at least one masking peptide does not bind to one or more CDRs of the activatable antibody.

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[0162] In some embodiments, the first masking peptide and the second masking peptide comprise leucine zipper coiled-coils. In some embodiments, the first masking peptide and the second masking peptide form parallel coiled-coils with high inter-coil affinities to reversibly impair antibody binding. In some embodiments, the activatable antibody inhibits antigen binding through steric hindrance, spatial occlusion, epitope adjacency, conformational restriction, or combinations thereof.

[0163] In some embodiments, the activatable antibody comprises a linker peptide linking the VH or VL with the cleavable peptide or linking the masking peptide with the cleavable peptide. In some embodiments, the linker peptide is comprised of an amino acid sequence of SEQ ID NOs: 536-538. In some embodiments, the linker peptide is comprised of about 1 to 30 amino acids. In some embodiments, the linker peptide is comprised of less than 30 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the linker peptide is a GlySer linker.

[0164] In some embodiments, the autoimmune or inflammatory disease is selected from type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, primary biliary cholangitis, lupus nephritis, Sjorgen’s syndrome- associated dry eye, and autoimmune optic neuritis. In some embodiments, the autoimmune or inflammatory disease is type 1 diabetes. In some embodiments, the autoimmune or inflammatory disease tissue is selected from pancreas, joint, intestine, brain, liver, kidneys, heart and eyes. In some embodiments, the autoimmune or inflammatory disease tissue is the pancreas. In some embodiments, the antibody is a humanized antibody, a chimeric antibody, or a human antibody.

[0165] In some or any of the foregoing aspects, the antibody is a humanized antibody, a chimeric antibody, or a human antibody. In some aspects, a nucleic acid encodes the activatable antibody of some or any of the foregoing aspects. In some or any of the foregoing aspects, a vector comprises the nucleic acid. In some aspects, the disclosure provides a host cell comprising some or any of the foregoing aspects.

[0166] In some aspects, the disclosure provides a method of producing an activatable antibody, comprising culturing the host cell of some or any of the foregoing aspects under conditions that produce the activatable antibody.

[0167] In some aspects, the disclosure provides a pharmaceutical composition comprising the activatable antibody of some or any of the foregoing aspects and a pharmaceutically acceptable carrier.

[0168] In some aspects, the disclosure provides a kit comprising the activatable antibody of some or any of the foregoing aspects and instructions for use.

[0169] In some aspects, the disclosure provides a method of treating an autoimmune or inflammatory disease in a subject, comprising administering to the subject an activatable antibody or pharmaceutical composition of some or any of the foregoing aspects.

[0170] In some or any of the foregoing aspects, the activatable antibody or pharmaceutical composition is administered to the subject intravenously, intramuscularly, systemically, or locally.

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[0172] FH13277368.1Attorney Docket No: TGY-00125

[0173] In some aspects, the disclosure provides use of the activatable antibody or pharmaceutical composition of some or any of the foregoing aspects in the manufacture of a medicament for the treatment of an autoimmune or inflammatory disease.

[0174] BRIEF DESCRIPTION OF THE DRAWINGS FIGs. 1A-1B illustrates configurations of activatable antibodies of the disclosure with cleavable peptides linked to the variable heavy chain (FIG. 1A) or light chain (FIG. IB) of antibodies. Various arrangements (1-4) illustrate placement of masking peptides, cleavable peptides, and linker peptides. MP: masking peptide, LP: linker peptide, CP: cleavable peptide, BD: binding domain. (1): BD-LP-CP-LP-MP; (2): BD-LP-CP-MP; (3): BD-CP-LP-MP; (4) BD-CP-MP.

[0175] FIG. 2 provides a schematic of various mechanisms of an anti-CD3 mAb: A) coating and downregulation of CD3 / TCR, B) T-cell killing, and C) induction of regulatory T cells.

[0176] FIG. 3 is a schematic depiction of the production of a tissue-specific activatable antibody by engineering a masking peptide and linker to an unmodified antibody.

[0177] FIGs. 4A-4C provides a schematic showing rationale for the activatable antibodies of the disclosure. FIG. 4A shows a representative unmodified antibody active in all tissues, while FIG. 4B shows conditional activation only in diseased tissues. FIG 4C shows representation of a methodology for testing conditional activation in the presence of different enzymes or in disease tissue-conditioned media.

[0178] FIG. 5 is a schematic representation of a workflow for tissue-specific cleavable peptide design. A stepwise funnel workflow narrows candidates progressively from identifying proteases enriched in target tissues (Step 1) to generating and refining peptide sequences (Steps 2-3), optimizing for reduced immunogenicity (Step 4), and selecting ranked candidates for in vitro validation (Step 5). P= protease, T= tissue.

[0179] FIG.6 depicts a schematic of an activatable antibody therapeutic platform tailored to a broad array of autoimmune and inflammatory diseases.

[0180] FIG. 7 illustrates tissue-acti vable biologies enabling organ- specific precision, here exemplified by pancreas-targeted activation for therapeutic application in Type 1 Diabetes (right: The right LOCATION).

[0181] FIG.8 demonstrates ex vivo functional masking using TACT-modified anti-mouse CD3s 145-2C11 parental antibody (2C11) incubated with mouse T cells isolated from splenocytes of Balb / c female mice. Two antibody concentrations (250 ng / mL and 62 ng / mL) were tested. TACT-modified 2C11 variant (#1, #2, #3) contained the same trypsin-sensitive cleavable linker peptide conferring pancreas-restricted activation but incorporated different masking peptides. IFNy levels were quantified using the LegendPLEX assay (BioLegend). One representative of two independent experiments.

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[0183] FH13277368.1Attorney Docket No: TGY-00125

[0184] FIGs.9A-9E demonstrate in vivo functional masking using TACT-modified anti-mouse CD3s 145-2C11 parental antibody (2C11) in NOD female mice. FIG. 9A: experimental overview. FIG. 9B:

[0185] Histogram of TACT masking of 2C11 in peripheral blood CD8+T cells at 4 h following a single intravenous injection of 0.1 mg / kg of antibody. FIG. 9C: Quantification of percent (%) mCD3 / TCR modulation in CD8+ T cells. FIG. 9D: Histogram of TACT masking of 2C11 in peripheral blood CD4+T cells at 4 h following a single intravenous injection of 0.1 mg / kg of antibody. FIG. 9E: Quantification of percent (%) mCD3 / TCR modulation in CD4+ T cells. CD3 / TCR modulation was assessed by flow cytometry.

[0186] FIGs. 10A-10C demonstrate in vivo safety improvement using TACT-modified anti-mouse CD3s 145-2C11 parental antibody (2C11) in NOD female mice. FIG. 10A: experimental schematic. FIG. 10B: quantification of cytokines (IFN, TNF, and IL-6) in the plasma of mice treated with unmasked anti-CD3e (2C11). FIG. 10C: quantification of cytokines (IFN, TNF, and IL-6) in the plasma of mice treated with TACT-masked anti-CD3e (2C11). Cytokines were measured in peripheral blood 4 h after a single 0.1 mg / kg injection using the LegendPLEX kit (BioLegend).

[0187] FIGs. 11A-11D demonstrate ex vivo functional masking using TACT-modified anti-mouse CD3s 145-2C11 parental antibody (2C11) incubated with mouse T cells for 24 hours. FIG. HA: Percent (%) CD3 / TCR modulation of CD4+ T cells from Balb / c female mice incubated with unmasked or TACT-masked 2C11. FIG. 11B: Percent (%) CD3 / TCR modulation of CD8+ T cells from Balb / c female mice incubated with unmasked or TACT-masked 2C11. FIG. 11C: Percent (%) CD3 / TCR modulation of CD4+ T cells from NOD female mice incubated with unmasked or TACT-masked 2C11. FIG. HD: Percent (%) CD3 / TCR modulation of CD8+ T cells from NOD female mice incubated with unmasked or TACT-masked 2C11. CD3 / TCR modulation was measured by flow cytometry.

[0188] FIGs.12A-12D demonstrate ex vivo trypsin-dependent selective functional activation using TACT-modified anti-mouse CD3s 145-2C11 parental antibody (2C11) incubated with mouse T cells. FIG. 12A:

[0189] Percent (%) CD3 / TCR modulation of CD4+ T cells from Balb / c female mice incubated with unmasked or TACT-masked 2C11 pre-incubated with trypsin. FIG. 12B: Percent (%) CD3 / TCR modulation of CD8+ T cells from Balb / c female mice incubated with unmasked or TACT-masked 2C11 pre-incubated with trypsin.

[0190] FIG. 12C: Percent (%) CD3 / TCR modulation of CD4+ T cells from NOD female mice incubated with unmasked or TACT-masked 2C11 pre-incubated with trypsin. FIG. 12D: Percent (%) CD3 / TCR modulation of CD8+ T cells from NOD female mice incubated with unmasked or TACT-masked 2C11 preincubated with trypsin. CD3 / TCR levels were analyzed by flow cytometry.

[0191] FIGs. 13A-13E demonstrate ex vivo diabetic pancreas-selecti ve functional activation using TACT-modified anti-mouse CD3s 145-2C11 parental antibody (2C11) incubated with mouse T cells. FIG. 13A: image of pancreas extracts from NOD diabetic mice. FIG. 13B: Percent (%) CD3 / TCR modulation of CD4+ T cells from Balb / c female mice incubated with unmasked or TACT-masked 2C11 in the presence of 14

[0192] FH13277368.1Attorney Docket No: TGY-00125

[0193] pancreas extracts. FIG. 13C: Percent (%) CD3 / TCR modulation of CD8+ T cells from Balb / c female mice incubated with unmasked or TACT-masked 2C11 in the presence of pancreas extracts. FIG. 13D: Percent (%) CD3 / TCR modulation of CD4+ T cells from NOD female mice incubated with unmasked or TACT-masked 2C11 in the presence of pancreas extracts. FIG. 13E: Percent (%) CD3 / TCR modulation of CD8+ T cells from NOD female mice incubated with unmasked or TACT-masked 2C 11 in the presence of pancreas extracts.

[0194] FIGs. 14A-14B demonstrate ex vivo CD3 / TCR modulation by the TACT-modified anti-human CD3s Teplizumab parental antibody (TZIELD) expressed in a mouse IgG2a backbone. FIG. 14A: Percent (%) CD3 / TCR modulation of human Jurkat T cells incubated with unmasked or TACT-masked TZIELD.

[0195] FIG. 14B: Percent (%) CD3 / TCR modulation of human primary T cells from a healthy donor incubated with unmasked or TACT-masked TZIELD. CD3 / TCR modulation was assessed by flow cytometry.

[0196] FIGs. 15A-15B demonstrate ex vivo T cell proliferation after incubation with the TACT-modified anti-human CD3s Teplizumab parental antibody (TZIELD) in a mouse IgG2a backbone incubated with T cells. FIG. 15A: Percent (%) divided human primary CD4+ T cells from a healthy donor incubated with unmasked or TACT-masked TZIELD. FIG. 15B: Percent (%) divided human primary CD8+ T cells from a healthy donor incubated with unmasked or TACT-masked TZIELD. Proliferation was quantified by flow cytometry as the percentage of CFSE-low (dividing) cells.

[0197] FIGs. 16A-16B demonstrate ex vivo MMP-9-dependent CD3 / TCR modulation using TACT-modified anti-human CD3s Teplizumab parental antibody (TZIELD) in a mouse IgG2a backbone. FIG.

[0198] 16A: Percent (%) CD3 / TCR modulation of human Jurkat T cells incubated with unmasked or TACT-masked TZIELD after pre-incubation with MMP-9. FIG. 16B: Percent (%) CD3 / TCR modulation of human primary T cells from a healthy donor incubated with unmasked or TACT-masked TZIELD after pre-incubation with MMP-9. Flow cytometry was used to assess CD3 / TCR modulation.

[0199] FIGs. 17A-17B demonstrate ex vivo MMP-9-dependent T cell proliferation using TACT-modified anti-human CD3s Teplizumab parental antibody (TZIELD) in a mouse IgG2a backbone incubated with primary human T cells. FIG. 17A: Percent (%) divided human primary CD4+ T cells incubated with unmasked or TACT-masked TZIELD after pre-incubation with MMP-9. FIG. 17B:

[0200] Percent (%) divided human primary CD8+ T cells incubated with unmasked or TACT-masked TZIELD after pre-incubation with MMP-9.

[0201] FIG. 18 is a schematic demonstrating a representative activatable antibody construct comprising the i nflammation regulating peptide linked to the Fc domain of the activatable antibody.

[0202] FIG. 19 shows representative activatable antibody structures for TACT1-4. TACT1 is a single linear peptide (SEQ ID NO: 545) linked via a trypsin-cleavable linker (SEQ ID NO: 548) to the VH domain of anti-CD3a 145-2C11. TACT2 is a single circular peptide (SEQ ID NO: 546) linked via a 15

[0203] FH13277368.1Attorney Docket No: TGY-00125

[0204] trypsin-cleavable linker (SEQ ID NO: 548) to the VH domain of anti-CD3s 145-2C11. The CDR sequences of the anti-CD3s 2C11 antibody are provided in Table 16A. TACT3 comprises two identical leucine zippers (SEQ ID NO: 547) that are linked via a trypsin-cleavable linker (SEQ ID NO: 548) to the VH domain and the VL domain of anti-CD3s 145-2C11. TACT4 is a single circular peptide (SEQ ID NO: 551) linked via an MMP-9 cleavable linker (SEQ ID NO: 552) to the VH domain of Teplizumab (TZIELD). CDR sequences of the anti-human CD3ε antibody TZIELD are provided in Table 20A.

[0205] DETAILED DESCRIPTION

[0206] The present disclosure provides tissue-specific activatable antibodies that bind immunomodulatory T cell molecules for the treatment of autoimmune and inflammatory conditions. The tissue-specific activatable antibodies of the disclosure target specific proteases present in autoimmune or inflammatory disease tissues to enable activation of the antibody in specific tissues and reduce systemic toxicity.

[0207] Definitions

[0208] The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

[0209] As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0210] The term “about” as used herein refers to the usual range understood by persons of ordinary skill and will vary up to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, "about" will mean up to plus or minus 10% of the particular value.

[0211] As used herein, the term “antibody” refers to a whole antibody comprising two light chain polypeptides and two heavy chain polypeptides. A heavy chain is typically comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) which defines the isotype of the immunoglobulin. The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. The term “immunoglobulin” as used herein is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four potentially inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. Within the structure of the immunoglobulin, the two heavy chains are inter-connected via disulfide bonds in the so-called “hinge region”. Each light chain is typically comprised of several regions; a light chain variable region

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[0214] (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions include regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and / or form of structurally defined loops), also termed complementarity-determining regions (CDRs), within regions that are typically conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino- terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0215] The term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a primatized antibody, a deimmunized antibody, and a fully human antibody. The antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, nonhuman primates, horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats and mice. The antibody can be a purified or recombinant antibody. In some embodiments, the antibody or antigen binding fragment thereof is a camelid heavy chain antibody, a nanobody, a humanized camelid heavy chain antibody, a bispecific humanized camelid heavy chain antibody, a camelid heavy chain antibody BiTE, a single variable domain, a monomer VHH domain, a dimer of VHH domains, a bispecific dimer of VHH domains, a dimer containing one VHH domain and a second functional domain, VHH-Fc, VHH-IgG, IgG-VHH, a knobs-in-holes assembly, a VHH-scFv, a IgG-VHH-scFv, or a Fc-VHH-scFv. In some embodiments, the antibody fragment is a camelid heavy chain antibody, a nanobody, a nanobody-HSA, a humanized camelid heavy chain antibody, a bispecific humanized camelid heavy chain antibody, a camelid heavy chain antibody BiTE, a single variable domain, a monomer

[0216] VHH domain, a dimer of VHH domains, a bispecific dimer of VHH domains, a dimer containing one VHH domain and a second functional domain, VHH-Fc, VHH-IgG, IgG-VHH, a knobs-in-holes assembly, a VHH-scFv, a IgG- VHH-scFv, or a Fc- VHH-scFv. Whole antibodies include antibody isotypes including IgM, IgG, IgA, IgD, and IgE antibodies.

[0217] As used herein, the term “human antibody” includes antibodies comprising variable and framework regions derived from human germline immunoglobulin sequences and a human immunoglobulin constant domain. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as a mouse, have been grafted onto human framework sequences.

[0218] As used herein, the term “humanized antibody” refers to a genetical I y engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to 17

[0219] FH13277368.1Attorney Docket No: TGY-00125

[0220] contain a high level of sequence identity to human variable domains. A humanized antibody can be obtained by grafting six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, into a homologous human acceptor framework region (FR). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e., the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.

[0221] An antibody comprises an Fc domain and an antigen-binding region. An antibody typically comprises two CH2-CH3 regions and a connecting region, e.g., a hinge region, e.g. at least an Fc domain. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant or “Fc” regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq, the first component in the classical pathway of complement activation. The Fc region of an immunoglobulin typically contains at least a CH2 domain and a CH3 domain of an immunoglobulin CH, and may comprise a connecting region, e.g., a hinge region. An Fc-region is typically in dimerized form via, e.g., disulfide bridges connecting the two hinge regions and / or non-co valent interactions between the two CH3 regions. The dimer may be a homodimer (where the two Fc region monomer amino acid sequences are identical) or a heterodimer (where the two Fc region monomer amino acid sequences differ in one or more amino acids). An Fc region- fragment of a full-length antibody can, for example, be generated by digestion of the full-length antibody with papain, as is well known in the art. An antibody may further comprise one or both of an immunoglobulin CHI region and a CL region.

[0222] As used herein, the term "monoclonal antibody," or “mAb” refers to an antibody which displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody which displays a single binding specificity and which has variable and optional constant regions derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

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[0225] The term “antibody binding region,” or “binding domain” refers to a region of the antigen, which comprises the epitope to which the antibody binds. An antibody binding region may be determined by epitope binding using biolayer interferometry, by alanine scan, or by shuffle assays (using antigen constructs in which regions of the antigen are exchanged with that of another species and determining whether the antibody still binds to the antigen or not). The amino acids within the antibody binding region that are involved in the interaction with the antibody may be determined by hydrogen / deuterium exchange mass spectrometry and by crystallography of the antibody bound to its antigen.

[0226] As used herein, the term “antigen-binding fragment” refers to a fragment of an antibody that retains the ability to bind a target antigen (e.g., CD3) and inhibit the activity of the target antigen. Such fragments include, e.g., a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, a Fab fragment, a Fab’ fragment, or an F(ab’)2 fragment. An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, triabodies, and diabodies are also included in the definition of antibody and are compatible for use in the methods described herein. In some embodiments, an antigenbinding fragment described herein comprises the CDRs of the light chain and heavy chain polypeptide of an antibody.

[0227] The terms “antigen” and “target” may, unless contradicted by the context, be used interchangeably in the context of the present invention.

[0228] The term “binding” as used herein refers to the binding of an antibody to a predetermined antigen or target, typically with a binding affinity corresponding to a KD of 1E6M or less, e.g., 5E7M or less, 1E7M or less, such as 5E8M or less, such as 1E8M or less, such as 5E9M or less, or such as 1E9M or less, when determined by biolayer interferometry using the antibody as the ligand and the antigen as the analyte and binds to the predetermined antigen with an affinity corresponding to a KD that is at least tenfold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.

[0229] The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, and is obtained by dividing kdby ka.

[0230] The term “kd” (sec-1), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. It is also referred to as the koffvalue or off-rate.

[0231] The term “k ” (M-1xsec-1), as used herein, refers to the association rate constant of a particular antibody-antigen i nteraction. It is also referred to as the konvalue or on-rate.

[0232] The term “epitope” means an antigenic determinant which is specifically bound by an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids, sugar side chains or a 19

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[0234] combination thereof and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues which are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked or covered by the antibody when it is bound to the antigen (in other words, the amino acid residue is within or closely adjacent to the footprint of the specific antibody).

[0235] As used herein, a “variant” refers to a protein or polypeptide sequence which differs in one or more amino acid residues from a parent or reference sequence. A variant may, for example, have a sequence identity of at least 80%, such as 90%, or 95%, or 97% or 99% identity to a parent or reference sequence. Also, or alternatively, a variant may differ from the parent sequence by 12 or less, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation(s) such as substitutions, insertions, or deletions of amino acid residues. As used herein, an "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different "replacement" amino acid residue. An "amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, larger "peptide insertions," can also be made, e.g. insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) may be naturally occurring or non- naturally occurring. An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0236] As used herein, a “variant antibody” or an “antibody variant,” used interchangeably herein, refers to an antibody that differs in one or more amino acid residues as compared to a parent or reference antibody, e.g., in the antigen-binding region, Fc-region or both. Likewise, a “variant Fc region” or “Fc region variant” refers to an Fc region that differs in one or more amino acid residues as compared to a parent or reference Fc region, optionally differing from the parent or reference Fc region amino acid sequence by 12 or less, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation(s) such as substitutions, insertions, or deletions of amino acid residues. The parent or reference Fc region is typically the Fc region of a human wild-type antibody which, depending on the context, may be a particular isotype. A variant Fc region may, in dimerized form, be a homodimer or heterodimer, e.g., where one of the amino acid sequences of the dimerized Fc region comprises a mutation while the other is identical to a parent or reference wild-type amino acid sequence.

[0237] As used herein, the term “agonistic antibody” binds the natural receptor to form a receptor antibody complex and which activates said receptor, thereby initiating a pathway signaling a further 20

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[0239] biological process. As used herein, the term “agonism” and agonistic” are used interchangeably to describe an antibody which is capable of, directly or indirectly, substantially inducing, promoting, or enhancing biological activity.

[0240] As used herein, the term “anti-idiotype antibody” refers to an antibody or antigen binding fragment that binds to the variable region of another antibody, which can include the antigen binding site.

[0241] As used herein, the term “activatable antibody” (e.g., anti-CD3 activatable antibody, also known as activatable anti-CD3 antibody) refers to an antibody or antigen binding fragment thereof with conditional activation in an autoimmune or inflammatory disease tissue. An activatable antibody generally comprises one or more binding domains (BD), one or more masking peptides (MP), one or more cleavable peptides (CP), and, optionally, one or more linker peptides (LP).

[0242] As used herein, “masking” refers to reducing target engagement of an antibody or

[0243] antigen-binding fragment through steric hindrance, spatial occlusion, epitope adjacency, conformational restriction, or combinations thereof. Masking may be achieved with a peptide, protein domain, antibody fragment, single-domain antibody, or other binding moiety and does not require specific binding to the idiotype, paratope, or complementarity-determining regions (CDRs) of the antibody, unless explicitly stated for particular embodiments (e.g., anti-idiotype masking embodiments).

[0244] As used herein, “activation” of an activatable antibody refers to restoration of target binding and / or restoration of an immunomodulatory function of the antibody following proteolytic cleavage of a cleavable peptide that removes, inactivates, or functionally disengages a masking element.

[0245] As used herein, “masking peptide” refers to an amino acid sequence which binds to one or more CDRs of the antibody or antigen binding fragment and inhibits the binding of the antibody or antigen binding fragment to an immunomodulatory T cell molecule. In some embodiments, the masking peptide can reduce target engagement of an antibody or antigen-binding fragment through steric hindrance, spatial occlusion, epitope adjacency, conformational restriction, or combinations thereof.

[0246] As used herein, “cleavable peptide” refers to an amino acid sequence that is selectively cleaved by enzymes in an autoimmune or inflammatory disease tissue.

[0247] As used herein, “linker peptide” refers to an amino acid sequence that links a masking peptide and the binding peptide and facilitates the controlled release of the masking peptide upon cleavage of the cleavable peptide.

[0248] The term “unmodified antibody” refers to an antibody prior to modification, such as the masking of an antibody with a masking peptide.

[0249] The term " T cell" refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells ( a.k.a. TH cells or CD4 + T cells) and subtypes, including THI,

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[0252] TH2, TH3, TH17, TH19, TFH cells, cytotoxic T cells (i.e., Tc cells, CD8+ T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells), regulatory T cells (a.k.a. Treg cells or suppressor T cells) and subtypes, including CD4+ FOXP3+ Tregcells, CD4+FOXP3- cells, Tri cells, Th3 cells, and Tregl7 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta T cells (y5T cells), including Vy9 / V52 T cells. Any one or more of the aforementioned or unmentioned T cells may be the target cell type for a method of use of the invention.

[0253] As used herein, the term " T cell activation" or "activation of T cells" refers to a cellular process in which mature T cells, which express antigen- specific T cell receptors on their surfaces, recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating or becoming competent to perform cell-based effector functions. T cell activation requires at least two signals to become fully activated. The first occurs after engagement of the T cell antigenspecific receptor (TCR) by the antigen-major histocompatibility complex (MHC), and the second by subsequent engagement of co-stimulatory molecules. These signals are transmitted to the nucleus and result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof.

[0254] As used herein, “Effector T cells” or “Teffs” or “Teff” refers to T lymphocytes that carry out a function of an immune response, such as killing tumor cells and / or activating an antitumor immune response which can result in clearance of the tumor cells from the body. Examples of Teff phenotypes include CD3+CD4+and CD3+CD8+. Teffsmay secrete, contain, or express markers such as IFNy, granzyme B, and TNFa. It is appreciated that Teffsmay not be fully restricted to these phenotypes.

[0255] As used herein, “Regulatory T cells” or “Tregs” or “Treg” refers to T lymphocytes that regulate the activity of other T cell(s) and / or other immune cells, usually by suppressing their activity. An example of a Treg phenotype is CD3+CD4+CD25+CD127dim. Tregsmay further express Foxp3. It is appreciated that may not be fully restricted to this phenotype.

[0256] As used herein, the term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid. Such analogs have modified R groups or modified peptide backbones but retain the same basic chemical structure as a naturally occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the 22

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[0258] one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0259] Substitution of one amino acid for another may be classified as a conservative or non-conservative substitution. In the context of the invention, a “conservative substitution” is a substitution of one amino acid with another amino acid having similar structural and / or chemical characteristics, such substitution of one amino acid residue for another amino acid residue of the same class.

[0260] In the context of the present invention, a substitution in an antibody is indicated as: Original amino acid — position — substituted amino acid;

[0261] Referring to the well-recognized nomenclature for amino acids, the three-letter code, or one letter code, is used, including the codes “Xaa” or “X” to indicate any amino acid residue. Thus, Xaa or X may typically represent any of the 20 naturally occurring amino acids. The term “naturally occurring” as used herein refers to any one of the following amino acid residues; glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine. Accordingly, the notation “K409R” or “Lys409Arg” means, that the antibody comprises a substitution of lysine with arginine in amino acid position 409. Substitution of an amino acid at a given position to any other amino acid is referred to as: Original amino acid — position; or e.g. “K409.” Such designation may be used interchangeably in the context of the invention but have the same meaning and purpose.

[0262] Furthermore, the term “a substitution” embraces a substitution into any one or the other nineteen natural amino acids, or into other amino acids, such as non-natural amino acids. For example, a substitution of amino acid K in position 409 includes each of the following substitutions: 409A, 409C, 409D, 409E, 409F, 409G, 409H, 4091, 409L, 409M, 409N, 409Q, 409R, 409S, 409T, 409V, 409W, 409P, and 409Y. This is, by the way, equivalent to the designation 409X, wherein the X designates any amino acid other than the original amino acid. These substitutions may also be designated K409A, K409C, etc. or K409A, C, etc. or K409A / C / etc. The same applies by analogy to each and every position mentioned herein, to specifically include herein any one of such substitutions.

[0263] The term "antigen presenting cell" or " APC" is a cell that displays foreign antigen complexed with MHC on its surface. T cells recognize this complex using T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMC), monocytes (such as THP-1), B lymphoblastoid cells (such as C1R. A2, 1518 B-LCL) and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens either by phagocytosis or by receptor-mediated endocytosis.

[0264] The term "antigen presentation" refers to the process by which APCs capture antigens and enables their recognition by T cells, e.g., as a component of an MHC-I and / or MHC-II conjugate.

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[0267] As used herein, the term "apoptosis" refers to the process of programmed cell death that occurs in multicellular organisms (e.g. humans). The highly-regulated biochemical and molecular events that result in apoptosis can lead to observable and characteristic morphological changes to a cell, including membrane blebbing, cell volume shrinkage, chromosomal DNA condensation and fragmentation, and mRNA decay. A common method to identify cells, including T cells undergoing apoptosis is to expose cells to a fluorophore-conjugated protein (Annexin V). Annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine on the outer leaflet of the plasma membrane, which is an early indicator that the cell is undergoing the process of apoptosis.

[0268] As used herein, the terms " Fc-mediated effector functions" or " Fc effector functions" refer to the biological activities of an antibody other than the antibody’s primary function and purpose. For example, the effector functions of a therapeutic agnostic antibody are the biological activities other than the activation of the target protein or pathway. Examples of antibody effector functions include Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); lack of activation of platelets that express Fc receptor; and B cell activation. Many effector functions begin with Fc binding to an Fey receptor.

[0269] As used herein, the term " Fc receptor" refers to a polypeptide found on the surface of immune effector cells, which is bound by the Fc region of an antibody. In some embodiments, the Fc receptor is an Fey receptor. There are three subclasses of Fey receptors, FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16). All four IgG isotypes (IgG 1, IgG2, IgG3 and IgG4) bind and activate Fc receptors FcyRI, FcyRIIA and FcyRIIIA. FcyRIIB is an inhibitory receptor, and therefore antibody binding to this receptor does not activate complement and cellular responses. FcyRI is a high affinity receptor that binds to IgG in monomeric form, whereas FcyRIIA and FcyRIIA are low affinity receptors that bind IgG only in multimeric form and have slightly lower affinity. The binding of an antibody to an Fc receptor and / or Clq is governed by specific residues or domains within the Fc regions. Binding also depends on residues located within the hinge region and within the CH2 portion of the antibody. In some embodiments, the agonistic and / or therapeutic activity of the antibodies described herein is dependent on binding of the Fc region to the Fc receptor (e.g., FcyR). In some embodiments, the agonistic and / or therapeutic activity of the antibodies described herein is enhanced by binding of the Fc region to the Fc receptor (e.g., FcyR).

[0270] As used herein, the term “host cell” is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

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[0273] Recombinant host cells include, for example, CHO cells, HEK293 cells, Expi293F cells, PER. C6 cells, NSO cells, and lymphocytic cells, and prokaryotic cells such as E. coli and other eukaryotic hosts such as plant cells and fungi.

[0274] As used herein, the terms "effective amount" or "effective dose," or similar terms used herein are intended to mean an amount of an agent (e.g., an activatable anti-CD3 antibody or an antigen-binding fragment thereof) that will elicit the desired biological or medical response (e.g., an improvement in one or more symptoms of an autoimmune or inflammatory disease). The terms "treat," "treating," and "treatment," as used herein, refer to therapeutic or preventative measures described herein. The methods of "treatment" employ administration to a subject, in need of such treatment, a human antibody of the present disclosure, for example, a subject in need of an attenuated immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0275] 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. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0276] Activatable Antibodies and Antigen-Binding fragments Thereof

[0277] The present disclosure provides activatable antibodies or antigen binding fragment thereof with conditional activation specific to an autoimmune or inflammatory disease tissue. An activatable antibody of the disclosure generally comprises one or more binding domains (BD), one or more masking peptides (MP), one or more cleavable peptides (CP), and, optionally, one or more linker peptides (LP) (FIGs. 1A-1B). The BD specifically binds an immunomodulatory T cell molecule. The BD may comprise one or more variable heavy chain (VH) CDRs or one or more variable light chain (VL) CDRs, or both. The BD may include a VH and / or a VL region. The masking peptide binds to the BD and inhibits or alters binding to the immunomodulatory T cell molecule, thereby rendering the activatable antibody functionally inactive outside of the autoimmune or inflammatory disease tissue. The CP comprises an amino acid sequence that is a substrate for a protease in an autoimmune or inflammatory disease tissue and is cleaved by the protease in the disease tissue(s) rendering the activatable antibody functionally active in the autoimmune or inflammatory disease tissue. In some embodiments, the activatable antibody of the disclosure comprises more than one CP. The LP provides flexibility and spacing between the MP and BD.

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[0280] In some embodiments, the LP modulates the interaction between the MP and BD and facilitates the controlled release of the MP upon cleavage of the CP In some embodiments, the LP connects the MP to the CP or the BD directly, or both. In some embodiments, the activatable antibody comprises multiple LPs. In some embodiments, the activatable antibody comprises one of the following configuration: BD-LP-CP-LP-MP, BD-LP-CP-MP; BD-CP-LP-MP; or BD-CP-MP

[0281] In some embodiments, the masking peptide and cleavable peptide are positioned at the

[0282] N-terminus of a heavy chain variable domain (VH), the N-terminus of a light chain variable domain (VL), within a hinge-proximal region, or at other positions that enable masking in the non-cleaved state and restoration of function after cleavage. In some embodiments, linkers are used between masking peptide, cleavable peptide, and antibody domains to tune masking efficiency and cleavage accessibility.

[0283] In some embodiments, the activatable antibody is not a bispecific T cell engager (TCE). In some embodiments, the activatable antibody does not redirect T cells toward a second antigen-expressing cell to induce proximity-mediated cytotoxicity or immunological synapse formation. In some embodiments, the activatable antibody binds the antigen upon proteolytic removal or inactivation of the masking peptide. In some embodiments, the function of the activatable antibody does not comprise enzymatic release of a non-antibody payload via a self-immolative linker. In some embodiments, therapeutic activity of the activatable antibody does not require administration of (i) a separate cell expressing a chimeric receptor that binds the antibody after cleavage, or (ii) a separate bispecific antibody that binds the activatable antibody after cleavage.

[0284] In some embodiments, therapeutic activity of the activatable antibody arises from tissue-localized activation within the autoimmune or inflammatory target tissue and is not dependent on achieving a predefined cumulative systemic exposure level of active, unmasked antibody in circulation.

[0285] In some embodiments, the activatable antibody further comprises a second functional binding domain that modulates an immune regulatory pathway in addition to CD3 modulation. Such embodiments are configured to deliver complementary or synergistic immunomodulatory signals within an autoimmune or inflammatory target tissue following activation.

[0286] The second functional binding domain may be configured to agonize or antagonize an immune checkpoint, co-stimulatory pathway, inhibitory pathway, cytokine pathway, or tolerance-associated receptor. In such embodiments, the activatable antibody remains a non-redirecting immunomodulatory molecule and is not configured to redirect T cells toward a second antigen-expressing cell, induce proximity-mediated cytotoxicity, or function as a bispecific T-cell engager. In certain embodiments, the activatable antibody is not configured to redirect T cells toward a second antigen-expressing cell and does not require administration of a separate bridging reagent, engineered receptor, or separate bispecific molecule that binds the activatable antibody after cleavage to achieve therapeutic effect. Therapeutic 26

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[0288] benefit arises from tissue-localized activation and immunomodulation rather than proximity-mediated redirected cytotoxicity.

[0289] In some embodiments, both functional domains act locally within the target tissue following protease-mediated activation.

[0290] Immunomodulatory T cell Molecules

[0291] The present disclosure provides activatable antibodies which bind to an immunomodulatory T cell molecule and are specifically activated in an autoimmune or inflammatory disease tissue of interest. An immunomodulatory T cell molecule refers to a protein expressed on the surface of a T cell that modulates an immune response (e.g., stimulates or inhibits an immune response), such as an immune checkpoint, a cytokine receptor, a chemokine receptor, a co-stimulatory molecule, an adhesion molecule, and an integrin.

[0292] Immune checkpoints can inhibit the immune response by restricting T cell activation, differentiation and function. In some embodiments of the present disclosure, PD-1, PD-L1, GITR, VISTA or CTLA-4 comprise inhibitory immunomodulatory T cell molecules. PD-1 / PD-L1 engagement prevents T cell activation by inhibiting downstream TCR-mediated signaling while CTLA-4 inhibits T cells by competing for ligand binding with the co-stimulatory molecule, CD28.

[0293] Cytokines and chemokines are small, secreted proteins that serve as mediators of cell to cell communication and can be broadly categorized as pro- or anti-inflammatory. In some embodiments of the present disclosure, the cytokine receptors, IL-2R, IL-7R, IL-10R, TNFR2, or chemokine receptors, CCR8, CCR4, or CXCR3 comprise immunomodulatory T cell molecules, wherein IL-R, IL-7 and IL-10 receptors regulate the development and survival of T cells, while CCR8 regulates cell trafficking.

[0294] Co-stimulatory molecules are cell surface receptors that amplify T cell signaling, and are generally members of the immunoglobulin superfamily (IgSF) or tumor necrosis factor receptor (TNFR) superfamily. In some embodiments of the present disclosure, CD28, ICOS, CD40 / CD40L, OX40 / OX40L, GITR, CD137, and TNFR2 comprise co-stimulatory immunomodulatory T cell molecules. In some embodiments of the present disclosure, CD28, ICOS, CD40 / CD40L, OX40 / OX40L, GITR, CD137, TNFR1, and TNFR2 comprise co-stimulatory immunomodulatory T cell molecules. CD28 and ICOS belong to the Ig family, while CD40, 0X40, GITR, and TNFR2 belong to the TNFR family.

[0295] Integrins such as ICAM-1 or adhesion molecules such as LFA-1 are immunomodulatory molecules which mediate T cell adhesion to antigen presenting cells and endothelial cells.

[0296] The activatable antibodies of the disclosure target one or more immunomodulatory T cell molecules as described herein for the treatment of autoimmune or inflammatory diseases.

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[0299] In some embodiments, the immunomodulatory T cell molecule is selected from a cytokine or chemokine receptor, an immune checkpoint, a member of the TNFR superfamily, a member of the IgSF, an integrin, or an adhesion molecule.

[0300] In some embodiments, the immunomodulatory T cell molecule is selected from one or more of the following: CD3, CD28, ICOS, IL-2Ra, IL-2R, IL-7R, IL-10R, PD-1, PD-L1, CTLA-4, TNFR2, CCR4, CCR8, CXCR3, 0X40, OX40L, GITR, TSLPR, CD137 (4-1BB), CD40L (CD154), LAG-3 (CD223), TIM-3 (HAVCR2), VISTA, TIGIT, GARP, LAP, NRP1, BTLA, CD27, CD39, CD47, CD73, CD276, 2B4 (CD244), αEβ7, CD62L, LFA-1, or ICAM-1, or a combination thereof. In some embodiments, the immunomodulatory T cell molecule is selected from one or more of the following: CD3, CD28, ICOS, IL-2, IL-2Ra, IL-2R, IL-7R, IL-10R, PD-1, PD-L1, CTLA-4, TNFR2, CCR4, CCR8, CXCR3, 0X40, OX40L, GITR, TSLPR, CD137 (4-1BB), CD40L (CD154), LAG-3 (CD223), TIM-3 (HAVCR2), VISTA, TIGIT, GARP, LAP, NRP1, BTLA, CD27, CD39, CD47, CD73, CD276, 2B4 (CD244), αEβ7, CD62L, LFA-1, or ICAM-1, or a combination thereof.

[0301] In some embodiments, binding of an activatable antibody of the disclosure to the immunomodulatory T cell molecule induces downregulation and / or functional inhibition of the CD3 / T Cell Receptor (TCR) complex. TCR and CD3 expression can be measured by flow cytometry, where reduced surface expression of CD3 reflects CD3 / TCR complex internalization.

[0302] In some embodiments, binding of an activatable antibody of the disclosure to the immunomodulatory T cell molecule activates effector T cells. T cell activation can be assessed by increased proliferation, CD25 (IL-2R) and CD69 expression, as well as cytokine production using flow cytometry or ELISA. In some embodiments, binding to the immunomodulatory T cell molecule induces TNFa and IFNy production. The percentage of proliferating and activated CD25+ or CD69+ cells within CD4+ and CD8+ cell subsets can be determined by flow cytometry. T-cell proliferation is determined as the percentage of CD4+ or CD8+ T cells with decreased Carboxyfluorescein Succinimidyl Ester (CFSE) signal.

[0303] In some embodiments, binding of an activatable antibody of the disclosure to the immunomodulatory T cell molecule activates regulatory T cells (Tregs). Tregactivation can be assessed by the expression of activation markers (CD25 and CD69), proliferation, or production of anti-inflammatory cytokines. In some embodiments, binding to the immunomodulatory T cell molecule induces TGFβ and IL-10 cytokine production, which can be measured by flow cytometry or ELISA. T-cell proliferation is determined as the percentage of CD4+ Tregswith decreased Carboxyfluorescein Succinimidyl Ester (CFSE) signal.

[0304] In some embodiments, binding of an activatable antibody of the disclosure to the immunomodulatory T cell molecule enhances T cell trafficking to the disease tissue. T cell trafficking can 28

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[0306] be measured in animal models ex vivo by assessing the total number and percent of T cells within a disease tissue using flow cytometry. T cell trafficking can refer to any T cell known in the art (e.g., Tregs, and CD4+ and CD8+ Teffs).

[0307] In some embodiments, binding of an activatable antibody of the disclosure to the immunomodulatory T cell molecule enhances immune tolerance. Immune tolerance can be measured by assessing the total number and percent of Tregswithin a tissue or expression of anti-inflammatory cytokines using flow cytometry. In murine models, enhanced immune tolerance can be assessed by resolution of disease burden (e.g., symptom severity, disease progression, and disease-specific biomarkers). In some embodiments, resolution of disease burden in a murine model of diabetes can be measured by insulitis severity and beta-cell mass preservation.

[0308] In some embodiments, the activatable antibody inhibits the immunomodulatory T cell molecule and / or activates Tregsand / or enhances immune suppression, and / or inhibits effector T cells. In some embodiments, the activatable antibody stimulates the immunomodulatory T cell molecule and / or activates Tregsand / or enhances immune suppression, and / or inhibits effector T cells.

[0309] In some embodiments, the activatable antibody inhibits interaction of the immunomodulatory T cell molecule with one or more endogenous ligands to and / or inhibits effector T cells.

[0310] In some embodiments, the immunomodulatory T cell molecule is expressed on Tregs, effector T cells, or both Tregsand effector T cells. T cells can be identified according to routine assays utilizing known surface markers by flow cytometry (e.g., Tregs(CD25+CD4+FoxP3+), Teffs(FoxP3-CD25-CD4+ / -CD8+ / -). In some embodiments, the T cells are murine T cells. In some embodiments, the T cells are human T cells. In some embodiments, the T cells are harvested from the blood, lymph node, or spleen of a mouse. In some embodiments, the T cells are isolated from PBMCs from a human subject.

[0311] In some embodiments, the immunomodulatory T cell molecule is a therapeutic target for the treatment of autoimmune or inflammatory diseases in a subject.

[0312] Masking Peptide

[0313] The present disclosure provides activatable antibodies that are comprised of at least one binding domain masking peptide. A binding domain masking peptide or “masking peptide” as provided herein refers to a peptide which binds to or interacts with one or more antigen binding domains (one or more CDRs) or one or more amino acids of one or more CDRs of the activatable antibody of the disclosure and inhibits binding to an immunomodulatory T cell molecule. In some embodiments, the masking peptide does not bind to one or more CDRs. When bound to or interacting with the antigen binding domain, the masking peptide blocks, inhibits, or otherwise prevents the ability of the binding domains to interact with the target immunomodulatory T cell molecule. In some embodiments, the 29

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[0315] masking peptide comprises an anti-idiotype antibody or fragment thereof which binds to the activatable antibody or fragment thereof to inhibit binding to an immunomodulatory T cell molecule. In some embodiments, the masking peptide does not comprise an anti-idiotype antibody. In some embodiments, the masking peptide reduces target engagement through steric hindrance, spatial occlusion, competitive epitope adjacency, conformational restriction, or a combination thereof. In some embodiments, the masking peptide does not bind to the idiotype, paratope, or CDRs of the antibody antigen binding site. In some embodiments, the masking peptide comprises a first masking peptide and a second masking peptide. In some embodiments, the first masking peptide and the second masking peptide are identical. In some embodiments, the first making peptide binds to the VH and the second masking peptide binds to the VL. In some embodiments, the masking peptide comprises two leucine zipper coiled-coils. In some embodiments, the two identical masking peptides form parallel heterodimeric coiled-coils with high intercoil affinities to reversibly impair antibody binding and biological function.

[0316] In some embodiments, the masking domain comprises a first masking peptide and a second masking peptide configured to associate with one another to form a multimeric masking assembly that sterically inhibits antigen binding in a target-agnostic manner.

[0317] In some embodiments, the first masking peptide and the second masking peptide are identical (homodimeric). In other embodiments, the first masking peptide and the second masking peptide are distinct (heterodimeric).

[0318] In some embodiments, the masking peptides associate through one or more of the following: (i) a-helical coiled-coil interactions, including but not limited to leucine zipper motifs;

[0319] (ii) P-strand or -sheet-forming interactions;

[0320] (iii) electrostatic or charge-complementary peptide interactions;

[0321] (iv) hydrophobic interface-mediated association;

[0322] (v) engineered peptide-peptide interaction domains, including consensus or de novo designed interaction motifs; or

[0323] (vi) covalent or semi-covalent reversible linkages, including disulfide-mediated, pH-sensitive, or protease-disruptable associations.

[0324] In some embodiments, the masking peptides are linked in-frame to distinct regions of the antibody, including the variable heavy (VH) domain and variable light (VL) domain, such that association of the masking peptides sterically obstructs antigen engagement prior to protease-dependent activation.

[0325] In some embodiments, association of the masking peptides is reversible or disrupted upon cleavage of a protease-cleavable linker, thereby restoring antigen-binding activity selectively within diseased tissue microenvironments. Leucine zipper-based masking peptides are provided as representative proof-of-concept embodiments and are not required for implementation of the universal masking

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[0328] architecture. In some embodiments, the masking peptide is comprised of one, two, three, four, five or six masking peptides each binding to one, two, three, four, five or six of the CDRs of the activatable antibody to inhibit binding of the light chain variable region and the heavy chain variable region to an immunomodulatory T cell molecule.

[0329] Cleavable Peptide

[0330] The activatable antibodies of the disclosure comprise at least one cleavable peptide (CP). The CP comprises an amino acid sequence that is selectively cleaved by an enzyme in an autoimmune or inflammatory disease tissue. In some embodiments, the cleavable peptide is selected such that the activatable antibody exhibits reduced cleavage and reduced activation in circulation (e.g., serum or plasma) relative to an autoimmune or inflammatory target tissue environment. This selective activation can reduce systemic exposure to unmasked active antibody while enabling restoration of target binding and immunomodulatory activity within the target tissue. In some embodiments, the disease tissue is selected from pancreas, joint, intestine, brain, liver, and kidneys. In some embodiments, the cleavable peptide is a substrate for a protease in the disease tissue. In some embodiments, the cleavable peptide comprises a consensus amino acid sequence recognized by a protease in an autoimmune or inflammatory disease tissue. In some embodiments, the cleavable peptide is less than 50 amino acids, less than 30 amino acids, less than 20 amino acids or less than 10 amino acids. In some embodiments, the cleavable peptide comprises an amino acid sequence set forth in Table 9, Table 11, Table 13, or Table 15, or a variant thereof. In some embodiments, the cleavable peptide comprises an amino acid sequence set forth in Table 10, Table 12, Table 14, Table 16, Table 17 or Table 18, or a variant thereof. In some embodiments, the cleavable peptide comprises an amino acid sequence set forth in SEQ ID NOs: 192-435 or a variant thereof.

[0331] In some embodiments, the protease is selected based on enriched, dysregulated, or disease-associated proteolytic activity within the autoimmune or inflammatory target tissue. In some embodiments, the disease tissue comprises pancreas, and the cleavable peptide is selected as a substrate for pancreatic serine proteases, including trypsin or trypsin-like proteases, which are enriched in pancreatic tissue and further elevated or dysregulated in inflammatory or autoimmune pancreatic disease. In some embodiments, the cleavable peptide enables preferential activation of the activatable antibody within pancreatic tissue relative to systemic circulation.

[0332] In some embodiments, the disease tissue comprises joint or synovial tissue, and the cleavable peptide is selected as a substrate for proteases enriched in inflamed joints, including aggrecanases or matrix-associated metalloproteases. In some embodiments, the protease comprises AD AMTS family

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[0335] members, including ADAMTS-5, which is upregulated in inflammatory joint disease and contributes to extracellular matrix remodeling within synovial tissue.

[0336] In some embodiments, the cleavable peptide is rationally designed based on known protease substrate preferences, disease-associated protease expression profiles, or proteolytic activity measurements in diseased tissue, while maintaining reduced susceptibility to cleavage in circulation.

[0337] In some embodiments, identification of a specific protease is not required, and the cleavable peptide is selected based on preferential cleavage observed in disease tissue extracts, conditioned media, or in vivo disease models relative to healthy tissue or systemic compartments.

[0338] Linker Peptide

[0339] In some embodiments, the activatable antibody of the disclosure comprises a linker peptide (LP). The LP operably links the MP and BD. In some embodiments, the LP provides flexibility and spacing between the MP and BD. In some embodiments, the LP modulates the interaction between the MP and BD and facilitates the controlled release of the MP upon cleavage of the CP. In some embodiments, the linker peptide links the cleavable peptide to the VH domain. In some embodiments, the linker peptide links the cleavable peptide to the VL domain. The LP is comprised of 50 amino acids or less. In some embodiments, the LP comprises 1 to 30 amino acids. In some embodiments, the LP comprises less than 30 amino acids, less than 20 amino acids, less than 10 amino acids or less than 5 amino acids. In some embodiments, the LP is comprised of 5 amino acids. In some embodiments, the LP is comprised of 10 amino acids. In some embodiments, the LP is comprised of 15 amino acids. In some embodiments, the LP is comprised of 20 amino acids. In some embodiments, the LP is comprised of 25 amino acids. In some embodiments, the linker is a GlySer linker. In some embodiments, the linker peptide is comprised of an amino acid sequence of SEQ ID NOs: 436-438.

[0340] The activatable antibody of the disclosure is engineered to reduce potential systemic toxicity by restricting activation within the tissue of interest for the treatment of autoimmune or inflammatory diseases.

[0341] Anti-CD3 Activatable Antibodies and Antigen-Binding fragments Thereof

[0342] The present disclosure provides activatable antibodies or antigen binding fragments thereof which specifically bind CD3 epsilon (NCBI Gene ID: 916). CD3 is a protein complex and T cell co-receptor that is involved in activating both CD4+ and CD8+ naive T cells. CD3 is comprised of four chains- CD3y, CD35, CD3s, and CD3^- that associate with the T cell receptor (TCR) and mediate activation through intracellular signaling cascades. Because of the ability for CD3 to modulate T cell responses, anti-CD3s monoclonal antibodies (mAbs) have been widely used in the treatment of inflammatory conditions and organ transplantation (Keam S. J. (2023). Drugs, 83(5), 439-445; Chatenoud L. (2003) Nature reviews.

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[0345] Immunology, 3(2), 123-132). In some embodiments, the activatable anti-CD3 antibody of the disclosure specifically bind the CD3s chain of the CD3 / TCR complex. Therefore, the term “CD3” as provided herein includes any of the recombinant or naturally-occurring forms of CD3 epsilon (CD3ε) or variants or homologs thereof.

[0346] Clinical development of anti-CD3 therapies has been limited by fundamental technical constraints inherent to systemic CD3 modulation. Specifically, systemic administration of anti-CD3 antibodies is associated with cytokine release syndrome, dose-limiting toxicities, transient efficacy, and the inability to safely sustain dosing over time (Chatenoud et al., Nature Reviews Immunology, 2007; PMID: 17641665; Utset et al., Journal of Rheumatology, 2002; PMID: 12233885). Efforts to mitigate toxicity through Fc receptor non-binding mutations improved tolerability but simultaneously reduced therapeutic potency, particularly the ability to eliminate pathogenic effector T cells within diseased tissues.

[0347] Accordingly, while anti-CD3 antibodies have repeatedly demonstrated powerful immunomodulatory and disease-modifying potential across autoimmune and inflammatory indications, their clinical utility has remained constrained by the absence of technologies enabling localized, tissue-restricted activation. Without being bound by theory, the activatable antibody disclosed herein maintains systemic inactivity of anti-CD3 antibodies while permitting conditional activation selectively within diseased tissues. In some embodiments, the activatable anti-CD3 antibodies of the disclosure are substantially functionally inert in circulation in the absence of the tissue-specific protease. Without being bound by theory, the anti-CD3 antibodies of the disclosure are useful in mitigating toxicities associated with current approaches.

[0348] In some embodiments, an activatable anti-CD3 antibody or antigen binding fragment of the disclosure is immunosuppressive and / or immunomodulatory. In some embodiments, an activatable anti-CD3 antibody or antigen binding fragment of the disclosure prevents antigen signaling through blockade of the CD3 / T cell receptor (TCR) resulting in internalization and activation-induced cell death (AICD), antibody-dependent cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC) of pathogenic effectors. Effector cell death leads to the induction of regulatory T cells (Tregs) which are important for long-term immune regulation (FIG. 2). The activatable anti-CD3 antibody or antigen-binding fragment is effective for blockade of TCR-mediated signaling, the Fc domain is involved in downstream killing of effectors and induction of Tregs through the Fc receptor (FcyR). Although CD3 is expressed on all T cells, highly activated and pathogenic T effector cells exhibit greater sensitivity to anti-CD3-induced cell death than regulatory T cells (Tregs). Thus, the activatable anti-CD3 antibodies of the disclosure are effective therapeutics due to the dual mechanism of action: short-term immunosuppression through CD3 / TCR internalization and effector T cell depletion and long-term immunomodulation through Treg induction

[0349] 33

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[0351] (Perruche, S., et al (2008). Nature Medicine, 14(5), 528-535; Belghith, M. et al (2003). Nature Medicine, 9(9), 1202-1208; You, S. et al (2007). PNAS, 104(15), 6335-6340).

[0352] In some embodiments, an activable anti-CD3 antibody of the disclosure comprises an engineered Fc non-binding (FNB) mutation(s)to reduce effector functions and avoid FcyR-mediated immune pathology. Such engineered FNB mAbs may have reduced systemic side effects, including reduced cytokine release syndrome, due to reduced interaction with Fc receptors (FcyR).

[0353] Importantly, the mechanism of anti-CD3 mAbs of the present disclosure in autoimmune and inflammatory disease is distinct from their use in oncology, where anti-CD3 mAbs are used as bispecific T cell engagers to activate T cells for cancer cell killing. In the context of autoimmune and inflammatory diseases, anti-CD3 mAbs function in an immunosuppressive and immunoregulatory capacity, whereas in oncology, they are used to promote immune activation. The activatable antibodies of the present disclosure are useful in the treatment of autoimmune and inflammatory diseases, while mitigating toxicity. The present disclosure provides activatable antibodies specific for immunomodulatory T cell molecules such as CD3 which are conditionally active in a disease tissue in a subject with an autoimmune or inflammatory condition (FIG. 3).

[0354] In some embodiments, the present disclosure provides activatable anti-CD3 antibodies or antigen binding fragments thereof, comprised of one or more binding domains that specifically bind CD3 (e.g., human CD3), one or more CD3 binding domain masking peptides (e.g., a human CD3 binding domain masking peptide), one or more cleavable peptides, and, optionally, one or more linker peptides. In some embodiments, the activatable anti-CD3 antibody is an activatable anti-CD3 antibody or antigen-binding fragment thereof.

[0355] In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure induces downregulation or inhibition of the CD3 / T Cell Receptor (TCR) complex. In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure induces T cell death. In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure induces the expansion and proliferation of regulatory T cells (Tregs). In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure induces or enhances Tregcytokine production. In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure induces any combination of the above properties. In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure induces T cell death in a subject with an autoimmune or inflammatory condition. In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure induces the expansion and proliferation of regulatory T cells (Tregs) in a subject with an autoimmune or inflammatory condition. In some

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[0358] embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure induces the production of TGF, IL- 10, or any combination thereof.

[0359] In some embodiments, the present disclosure provides use of an activatable anti-CD3 antibody in the treatment of an autoimmune or inflammatory disease by administering the activatable antibody or a pharmaceutical composition comprising the antibody to a subject. In some embodiments, the subject has an autoimmune or inflammatory disease selected from type 1 diabetes (T1D), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis (AD), multiple sclerosis (MS), Sjorgen’s syndrome-associated dry eye, and autoimmune optic neuritis. Extensive preclinical clinical evidence demonstrates that pathogenic T cells and CD3-mediated signaling play a central role in the initiation, persistence, and recurrence of multiple autoimmune and inflammatory diseases, including:

[0360] Type 1 diabetes (Herold et al., New England Journal of Medicine, 2002; Belghith et al., Nature Medicine, 2003)

[0361] Inflammatory bowel disease (Ludviksson et al., Journal of Immunology, 1997) Crohn’s Disease (D’Haens & Daperno, Current Gastroenterology Reports, 2006) Multiple Sclerosis (Tran et al., International Immunology, 2001; Kohm et al., Journal of Immunology, 2005; Ochi et al., Nature Medicine, 2006)

[0362] Rheumatoid Arthritis (Hughes et al., Journal of Immunology, 1994)

[0363] Psoriatic Arthritis (Utset et al., Journal of Rheumatology, 2002)

[0364] Graft-versus-Host Disease (Blazar et al., Journal of Immunology, 1997; Carpenter et al., Blood, 2002)

[0365] Arthritis Chronicity (Chang et al., Cell Reports, 2021)

[0366] Anti-CD3 antibodies have shown robust efficacy across diverse T-cell-dependent disease models, including experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis, inflammatory colitis, and transplant rejection, establishing CD3 as a broadly disease-relevant immunomodulatory target across organ systems (Chatenoud et al., Nature Reviews Immunology, 2007; PMID: 17641665).

[0367] In some embodiments, the present disclosure provides methods of treating a subject with type 1 diabetes comprising administering the activatable anti-CD3 antibody or antigen binding fragment thereof.

[0368] In some embodiments, the present disclosure provides methods of treating a subject with inflammatory bowel disease, comprising administering the activatable anti-CD3 antibody or antigen binding fragment thereof. In inflammatory bowel disease models, administration of anti-CD3 antibodies suppresses T-cell-driven colitis and promotes regulatory immune responses, including TGF-P-dependent tolerance induction in TNP-KLH-induced colitis (Ludviksson et al., Journal of Immunology, 1997;

[0369] PMID: 9317162). In the clinical setting, humanized anti-CD3 antibodies such as visilizumab have demonstrated biological and clinical activity in Crohn’s disease, further validating CD3 signaling as a 35

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[0371] therapeutic axis in intestinal inflammation (D’Haens and Daperno, Current Gastroenterology Reports, 2006; PMID: 17105690).

[0372] In some embodiments, the present disclosure provides methods of treating a subject with multiple sclerosis comprising administering the activatable anti-CD3 antibody or antigen binding fragment thereof. In neuroinflammatory disease, multiple studies in EAE models demonstrate that non-mitogenic, nondepleting anti-CD3 antibodies reverse established disease, induce T-cell unresponsiveness, and promote regulatory CD4+T-cell populations (Tran et al., International Immunology, 2001; PMID: 11526091; Kohm et al., Journal of Immunology, 2005; PMID: 15814673; Ochi et al., Nature Medicine, 2006; PMID: 16715091). These findings highlight the potent disease-modifying capacity of CD3 modulation in T-cell-mediated neuroinflammation.

[0373] In some embodiments, the present disclosure provides methods of treating a subject with rheumatoid arthritis comprising administering the activatable anti-CD3 antibody or antigen binding fragment thereof. In inflammatory arthritis, including rheumatoid arthritis and psoriatic arthritis, anti-CD3 antibodies have demonstrated efficacy in both preclinical and clinical settings. In collagen-induced arthritis models, non-mitogenic anti-CD3 therapy induces T-helper cell hyporesponsiveness and ameliorates disease (Hughes et al., Journal of Immunology, 1994; PMID: 8089501). In humans, a Phase I / II clinical trial of a humanized Fc receptor-nonbinding anti-CD3 antibody (teplizumab; huOKT3yl(Ala-Ala)) in patients with psoriatic arthritis resulted in rapid and substantial clinical improvement, with >75% reduction in inflamed joints in most patients at Day 30 (Utset et al., Journal of Rheumatology, 2002; PMID: 12233885). These results provided direct clinical validation that pathogenic T cells and CD3 signaling are disease-relevant targets in inflammatory arthritis.

[0374] In addition, emerging mechanistic studies in inflammatory arthritis demonstrate that disease chronicity and relapse are driven by long-lived tissue-resident memory T cells (TRM) that persist locally within inflamed joints during clinical remission. These synovial TRM populations exhibit restricted T-cell receptor repertoires, pro-inflammatory transcriptional signatures, and the capacity to nucleate jointspecific disease flares upon antigen re-exposure. Experimental depletion of synovial TRM cells during remission, achieved through localized diphtheria toxin-mediated ablation in animal models, prevents arthritis recurrence in a site-specific manner, providing direct proof that local T-cell elimination can disrupt disease chronicity (Chang et al., Cell Reports, 2021; PMID: 34706228). Notably, these studies underscore that selectively neutralizing or depleting pathogenic T cells within diseased tissues is therapeutically effective. In some embodiments, provided herein are anti-CD3 activatable monoclonal antibodies or antigen binding fragments thereof, comprising heavy and light chain variable sequences as set forth in Table 2.

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[0377] In some embodiments, the activatable anti-CD3 antibody is specific to human, murine, or primate CD3. In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure comprises the variable sequences of 0KT3 (Bluestone et al (1994) Methods and materials for modulation of the immunosuppressive activity and toxicity of monoclonal antibodies, US6491916B1, the contents of which are incorporated herein by reference). In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure comprises the variable sequences of Teplizumab (humanized 0KT3) wherein the light chain variable region comprises SEQ ID NO: 13 and the heavy chain variable region comprises SEQ ID NO: 14 (Ramos et al, 2023. NEJM, 389:2151-2161; Koenig et al (2006) Methods for the treatment of autoimmune disorders using immunosuppressive monoclonal antibodies with reduced toxicity US8663634B2, the contents of which are incorporated herein by reference). Teplizumab is a humanized version of murine OKT3, which retains the same antigen binding domains as OKT3 but with modifications of amino acids at positions 234 and 235 of the human IgGl Fc resulting in decreased Fc binding (Masharani and Becker, 2011, Expert Opin Biol Ther 10(3):459–465). In some embodiments, the anti-CD3 antibody is selected from oxtelixizumab and visilizumab.

[0378] It is well known in the art that mutations may be introduced into proteins having a well characterized sequence and three dimensional structure such as an antibody without the loss of function. Accordingly, in some embodiments, the activatable antibody or antigen binding fragment of the disclosure comprises a variant of the VL and / or VH as set forth in SEQ ID NOs: 13 and 14, respectively. In some embodiments, an activatable antibody or antigen binding fragment of the disclosure comprises a variant of the light chain CDR1-3 and heavy chain CDR1-3 as set forth in SEQ ID NOs: 1-12. The variant may differ in one or more amino acids as compared to the framework regions i.e. FR1, FR2, FR3 and FR4 of the parent VH and / or VL sequence, e.g., in one or more framework regions, but still allows the antigenbinding region to retain at least a substantial proportion (at least about 90 percent, 95 percent or more) or even retain all of the affinity and / or specificity of the parent antibody. Typically, such functional variants retain significant sequence identity to the parent sequence. Exemplary variants include those which differ from the respective parent VH or VL region by 5 or less, such as 5, 4, 3, 2 or 1 mutation(s) such as substitution of amino acid residues. Exemplary variants include those which differ from the VH and / or VL regions of the parent sequences mainly by conservative amino acid substitutions; for instance, 5, such as 5, 4, 3, 2 or 1 of the amino acid substitutions in the variant can be conservative. In a further aspect of the invention the antibody may comprise at most 1, 2 or 3 mutations in the VH framework regions and / or in the VL framework regions, respectively. Such mutations may be substitutions. It is preferred that such substitutions do not significantly change the binding affinity and / or binding specificity of the activatable antibodies of the invention.

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[0381] In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure comprises the heavy and light chain variable sequences of Teplizumab / 0KT3, comprising six CDRs comprising the sequence set forth in SEQ ID NOs: 1-6. (Norman 1995, Ther Drug Monit. (6):615-20). In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, or antigen binding fragment thereof wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6.

[0382] In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure comprises a heavy chain and a and light chain, wherein the heavy chain is set forth in SEQ ID NO: 546 and the light chain is set forth in SEQ ID NO: 545.

[0383] In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, or antigen binding fragment thereof wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12.

[0384] In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, or antigen binding fragment thereof wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 542, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 543, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 544; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 539, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 540, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 541.

[0385] In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure comprises a light chain and a heavy chain wherein the light chain comprises an amino acid sequence set forth in SEQ ID NO: 538 and the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 537.

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[0388] In some embodiments, the activatable anti-CD3 antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 531, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 532, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 533; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 534, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 535, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 536.

[0389] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises a CDR-1 molecule comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0390] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0391] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises a CDR-1 molecule comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.

[0392] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable 39

[0393] FH13277368.1Attorney Docket No: TGY-00125

[0394] antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises a CDR-1 molecule comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.

[0395] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises a CDR-2 molecule comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0396] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises a CDR-2 molecule comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5.

[0397] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises a CDR-2 molecule comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8.

[0398] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises a CDR-2 molecule 40

[0399] FH13277368.1Attorney Docket No: TGY-00125

[0400] comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11.

[0401] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises a CDR-3 molecule comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0402] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.

[0403] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises a CDR-3 molecule comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9.

[0404] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, a CDR-3

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[0407] molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12.

[0408] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises a variable light chain comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, a variable light chain comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, a variable light chain comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, a variable light chain comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13.

[0409] In some embodiments, the anti-CD3 activatable antibody or antigen binding fragment of the disclosure comprises one or more binding domains. In some embodiments, the anti-CD3 activatable antibody comprises a variable heavy chain comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, a variable heavy chain comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, a variable heavy chain comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, a variable heavy chain comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.

[0410] In some embodiments, the activatable antibody or antigen binding fragment of the disclosure comprises the variable domains of any of the following: Foralumab (Dean et al (2019) Combination therapies and methods using anti-CD3 modulating agents and anti-il6 antagonists, US20200069797A1, the contents of which are incorporated herein by reference), Otelixumab, Visilizumab (Tso et al (1996) Mutated nonactivating Igg2 domains and anti cd3 antibodies incorporating the same, US 5,834,597 the contents of which are incorporated herein by reference), or Tregalizumab.

[0411] TABLE 1: Anti-CD3 CDR Sequences

[0412] SEQ ID NO Description Sequence

[0413] 1 Teplizumab / OKT3 CDR-L1 SASSSVSYMN

[0414] 2 Teplizumab / OKT3 CDR-L2 DTSKLAS

[0415] 3 Teplizumab / OKT3 CDR-L3 QQWSSNPFT

[0416] 4 Teplizumab / OKT3 CDR-H1 RYTMH

[0417] 5 Teplizumab / OKT3 CDR-H2 YINPSRGYTNYNQKFKD

[0418] 6 Teplizumab / OKT3 CDR-H3 YYDDHYCLDY

[0419]

[0420] 7 Anti-hCD3ε #CH2527 CDR-L1 GSSTGAVTTSNYAN

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[0423] 8 Anti-hCD3ε #CH2527 CDR-L2 GTNKRAP

[0424] 9 Anti-hCD3ε #CH2527 CDR-L3 ALWYSNLWV

[0425] 10 Anti-hCD3ε #CH2527 CDR-H1 TYAMN

[0426] 11 Anti-hCD3ε #CH2527 CDR-H2 RIRSKYNNYATYYADSVKG

[0427] 12 Anti-hCD3ε #CH2527 CDR-H3 HGNFGNSYVSWFAY

[0428] 439 Anti-CD3ε, clone 145-2C11 CDR-H1 GFTFSGYGMHWV

[0429] 440 Anti-CD3ε, clone 145-2C11 CDR-H2 ITSSSINIKY

[0430] 441 Anti-CD3ε, clone 145-2C11 CDR-H3 ARFDWDKNYWGQG

[0431] 442 Anti-CD3ε, clone 145-2C11 CDR-L1 QASQDISNYLN

[0432] 443 Anti-CD3ε, clone 145-2C11 CDR-L2 YTNKLADGV

[0433] 444 Anti-CD3ε, clone 145-2C11 CDR-L3 QQYYNYPWT

[0434] 531 Anti-hCD3ε Foralumab CDR-L1 RASQSVSSYLA

[0435] 532 Anti-hCD3ε Foralumab CDR-L2 DASNRAT

[0436] 533 Anti-hCD3ε Foralumab CDR-L3 QQRSNWPPLT

[0437] 534 Anti-hCD3ε Foralumab CDR-H1 GYGMH

[0438] 535 Anti-hCD3ε Foralumab CDR-H2 VIWYDGSKKYYVDSVKG

[0439]

[0440] 536 Anti-hCD3ε Foralumab CDR-H3 QMGYWHFDL

[0441] TABLE 2: Anti-CD3 Variable Chain Sequences of OKT3 / Teplizumab

[0442] SEQ ID Description Sequence

[0443] NO

[0444] 13 OKT3yl Light MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCSAS Chain SSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTD YFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLIQITRVAAPSVFI CDR1 FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ CDR2 ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP CDR3 VTKSFNRGEC

[0445] 14 OKT3yl (alaMGWSCIILFLVATATGVHSQVQLVQSGGGVVQPGRSLRLSCKA ala) Heavy SGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFK Chain DRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDY WGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF CDR1 PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT CDR2 QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPS CDR3 VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0446]

[0447] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0448] Anti-PD-1 Activatable Antibodies and Antigen-Binding fragments Thereof

[0449] The present disclosure provides activatable antibodies or antigen binding fragments thereof which specifically bind programmed cell death protein-1 (PD-1) (NCBI Gene ID: 5133). PD-1 is a key immune checkpoint receptor expressed on activated T cells, acting to inhibit T cell function and proliferation. PD-1 ensures immune homeostasis by preventing excessive immune responses and limiting autoimmunity. However, excessive inhibition of the PD-1 pathway, as seen with the use of PD-1 inhibitors in cancer 43

[0450] FH13277368.1Attorney Docket No: TGY-00125

[0451] therapy, can lead to immune-related adverse events resembling autoimmune diseases. Conversely, a decrease in PD-1 signaling may exacerbate immune activation, contributing to autoimmune pathology. Unlike PD-1 inhibitors used in oncology, PD-1 agonists are being developed to suppress immune activity and restore tolerance in autoimmune diseases. This approach capitalizes on their ability to dampen T-cell activation. However, current PD-1 agonists suffer from significant limitations, primarily their systemic mechanism of action. This lack of tissue specificity poses challenges in efficacy and safety, particularly in diseases requiring localized immune modulation, such as rheumatoid arthritis, ulcerative colitis, and type 1 diabetes. PD-1 agonists depend on their ability to suppress hyperactive T cells directly in the inflamed tissue microenvironment. However, the systemic activity of current PD-1 agonists undermines this goal. In some embodiments, the activatable anti-PD- 1 antibody or antigen binding fragments thereof is comprised of one or more binding domains that specifically binds PD- 1, one or more PD- 1 binding domain masking peptides, one or more cleavable peptides, and, optionally one or more a linker peptides. In some embodiments, the activatable anti-PD- 1 antibody is an activatable anti-PD- 1 antibody or antigenbinding fragment thereof.

[0452] In autoimmune and inflammatory settings, augmenting PD-1 inhibitory signaling via agonist approaches has shown broad therapeutic promise across multiple disease contexts (Inhibitory receptor agonists: the future of autoimmune disease therapeutics?, Curr Opin Immunol, 2020; PMID: 32619929), including:

[0453] Multiple Sclerosis (Hirata et al.,. / . Immunol., 2007)

[0454] Systemic Lupus Erythematosus (Ding et al., Clin. Immunol., 2006)

[0455] Inflammatory Bowel Disease (Song et al., Gut, 2015)

[0456] Rheumatoid Arthritis (Raptopoulou et al., Arthritis Rheum., 2010)

[0457] Type 1 diabetes (Gao et al., Transplantation, 2003)

[0458] Consistent with this concept, reinforcing PD-1 / PD-L1 inhibitory signaling has demonstrated activity across diverse preclinical autoimmune models, including experimental autoimmune encephalomyelitis (EAE) (Hirata et al., J Immunol, 2007; PMID: 17202353), lupus-like disease in autoimmune BXSB mice (Ding et al., Clin Immunol, 2006; PMID: 16386962), inflammatory bowel disease models such as DSS colitis (Song et al., Gut, 2015; PMID: 24902766), and inflammatory arthritis models (Raptopoulou et al., Arthritis Rheum, 2010; PMID: 20506224; Wang et al., Rheumatol Int, 2011; PMID: 20035333). PD-1 pathway engagement has also supported durable graft outcomes in transplantation settings relevant to autoimmune disease, including islet allograft survival (Gao et al., Transplantation, 2003; PMID: 14508368). Collectively, these studies establish PD-1 agonism as a disease-modifying strategy across organ systems where pathogenic T-cell activity is central.

[0459] 44

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[0461] Despite this, current PD-1 agonist strategies are constrained by a predominantly systemic mechanism of action, which can undermine both efficacy and safety in diseases where the desired pharmacology is localized immune dampening within inflamed tissue microenvironments. Systemic PD-1 agonism can impose broad T-cell suppression outside the target lesion, creating a narrow therapeutic window and limiting dose escalation or treatment duration required for durable benefit. This limitation is particularly salient in disease settings where PD-1 signaling is downregulated locally at sites of inflammation, yet systemic exposure continues to dominate pharmacology and risk.

[0462] Without being bound by theory, the activatable anti-PD- 1 antibodies of the disclosure maintain reduced systemic activity while enabling conditional, site-specific activation of PD-1 agonist function within inflamed tissues, where hyperactive pathogenic T cells are concentrated and where therapeutic PD-1 signaling is most likely to restore local immune balance. By focusing PD-1 agonism to sites of diseaserelevant inflammation, activatable anti-PD- 1 antibodies have the potential to (i) increase effective potency at the lesion, (ii) expand the therapeutic window, and (iii) mitigate systemic immunosuppression liabilities, thereby enabling safer and more efficacious PD-1 agonist therapies for organ-specific autoimmune and inflammatory diseases.

[0463] In some embodiments, the activatable anti-PD- 1 antibody or antigen binding fragments thereof is used in the treatment of an autoimmune or inflammatory disease. In some embodiments, the anti-PD- 1 activatable antibodies are used for the treatment of type 1 diabetes (T1D), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis (AD), multiple sclerosis (MS), primary biliary cholangitis, lupus nephritis, Sjorgen’s syndrome-associated dry eye, and / or autoimmune optic neuritis.

[0464] In some embodiments, provided herein are anti-PD- 1 activatable monoclonal antibodies or antigen binding fragments thereof, comprising heavy and light chain variable sequences as set forth in Table 3.

[0465] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragments thereof comprises one or more binding domains that specifically bind PD-1. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR, wherein the at least one CDR comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20.

[0466] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragments thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable 45

[0467] FH13277368.1Attorney Docket No: TGY-00125

[0468] region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 21, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 22, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 24, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 25, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 26.

[0469] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragments thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 27, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 28, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 30, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32.

[0470] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragments thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38.

[0471] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragments thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 39, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 40, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 41; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 42, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 43, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44.

[0472] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragments thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 45, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 46, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 47; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 48, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 49, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 50.

[0473] 46

[0474] FH13277368.1Attorney Docket No: TGY-00125

[0475] In some embodiments, the anti-PD-1 activatable antibody or antigen binding fragments thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 51, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 52, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 53; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 54, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 55, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 56.

[0476] In some embodiments, the light chain variable CDR-1 region is comprised of the amino acid sequences set forth in SEQ ID NOs: 15, 21, 27, 33, 39, 45 or 51.

[0477] It is well known in the art that mutations may be introduced into proteins having a well characterized sequence and three dimensional structure such as an antibody without the loss of function. Accordingly, in some embodiments, the activatable antibody comprises a variant of the light chain CDR1-3 and heavy chain CDR1-3 as set forth in SEQ ID NOs: 15-56. The variant may differ in one or more amino acids as compared to the framework regions i.e. FR1, FR2, FR3 and FR4 of the parent VH and / or VL sequence, e.g., in one or more framework regions, but still allows the antigen-binding region to retain at least a substantial proportion (at least about 90 percent, 95 percent or more) or even retain all of the affinity and / or specificity of the parent antibody. Typically, such functional variants retain significant sequence identity to the parent sequence. Exemplary variants include those which differ from the respective parent VH or VL region by 5 or less, such as 5, 4, 3, 2 or 1 mutation(s) such as substitutions, of amino acid residues. Exemplary variants include those which differ from the VH and / or VL regions of the parent sequences mainly by conservative amino acid substitutions; for instance, 5, such as 5, 4, 3, 2 or 1 of the amino acid substitutions in the variant can be conservative. In a further aspect of the invention the antibody may comprise at most 1, 2 or 3 mutations in the VH framework regions and / or in the VL framework regions, respectively. Such mutations may be substitutions. It is preferred that such substitutions do not significantly change the binding affinity and / or binding specificity of the activatable antibodies of the invention.

[0478] In some embodiments, the anti-PD-1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.

[0479] 47

[0480] FH13277368.1Attorney Docket No: TGY-00125

[0481] In some embodiments, the anti-PD-1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21.

[0482] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27.

[0483] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 33.

[0484] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39.

[0485] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody 48

[0486] FH13277368.1Attorney Docket No: TGY-00125

[0487] comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 45.

[0488] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51.

[0489] In some embodiments, the heavy chain variable CDR-1 region is comprised of the amino acid sequences set forth in SEQ ID NOs: 18, 24, 30, 36, 42, 48, or 54.

[0490] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.

[0491] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24.

[0492] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody 49

[0493] FH13277368.1Attorney Docket No: TGY-00125

[0494] comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30.

[0495] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36.

[0496] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 42.

[0497] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, a CDR-1 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48.

[0498] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-1, wherein the at least one CDR-1 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, a CDR-1 molecule 50

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[0500] comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, a CDR-1 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, a CDR-1 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54.

[0501] In some embodiments, the light chain variable CDR-2 region is comprised of the amino acid sequences set forth in SEQ ID NOs: 16, 22, 28, 34, 40, 46 or 52.

[0502] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16.

[0503] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22.

[0504] In some embodiments the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28.

[0505] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, a CDR-2 molecule 51

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[0507] comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 34.

[0508] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40.

[0509] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 46.

[0510] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52.

[0511] In some embodiments, the heavy chain variable CDR-2 region is comprised of the amino acid sequences set forth in SEQ ID NOs: 19, 25, 31, 37, 43, 49, or 55.

[0512] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, a CDR-2 molecule 52

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[0514] comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19.

[0515] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25.

[0516] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 31.

[0517] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37.

[0518] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set 53

[0519] FH13277368.1Attorney Docket No: TGY-00125

[0520] forth in SEQ ID NO: 43. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 43.

[0521] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 49.

[0522] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-2, wherein the at least one CDR-2 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, a CDR-2 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, a CDR-2 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, a CDR-2 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55.

[0523] In some embodiments, the light chain variable CDR-3 region is comprised of the amino acid sequences set forth in SEQ ID NOs: 17, 23, 29, 35, 41, 47, or 53.

[0524] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0525] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set 54

[0526] FH13277368.1Attorney Docket No: TGY-00125

[0527] forth in SEQ ID NO: 23. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23.

[0528] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 29.

[0529] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35.

[0530] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41.

[0531] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD- 1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47.

[0532] 55

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[0534] In some embodiments, the anti-PD-1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53.

[0535] In some embodiments, the heavy chain variable CDR-3 region is comprised of the amino acid sequences set forth in SEQ ID NOs: 20, 26, 32, 38, 44, 50 or 56.

[0536] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20.

[0537] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26.

[0538] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32.

[0539] 56

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[0541] In some embodiments, the anti-PD-1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 38.

[0542] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 44.

[0543] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50.

[0544] In some embodiments, the anti-PD- 1 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-PD-1 activatable antibody comprises at least one CDR-3, wherein the at least one CDR-3 comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, a CDR-3 molecule comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, a CDR-3 molecule comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, a CDR-3 molecule comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56.

[0545] In some embodiments, the anti-PD-1 activatable antibody or antigen binding fragment thereof comprises the variable domains of any one of the following: JNJ-4703; Anaptys Bio Clone #3.7C6 and 57

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[0547] Clone #437M5 (Kehry et al (2020) Pd-1 agonist and methods of using same, WO2020247648A2 incorporated herein by reference; Kehry et al (2020) Pd-1 agonist and methods of using same, US20220235132A1, incorporated herein by reference), Clone #PDlAB-6; CC-90006 (Bennett et al (2016) Pd-1 binding proteins and methods of use thereof, US20170088618A1, incorporated herein by reference); PT627 (Higginson-Scott et al (2021) Multi-paratopic anti-pd-1 antibodies and uses thereof, US20240052034A1 incorporated herein by reference); RubrYc Therapeutics: RTX-002.

[0548] TABLE 3: Anti-PD-1 CDR Sequences

[0549] SEQ ID Name Description Sequence

[0550] NO

[0551] 15 Rosnilimab (Anaptys Bio, CDR-L1.1 TASSSVSSSYFH

[0552] 16 US20220235132)* CDR-L2.1 STSNLAS

[0553] 17 CDR-L3.1 HQYHRSPLT

[0554] 18 CDR-H1.1 DYSMH

[0555] 19 CDR-H2.1 WINIETYYPTYADQFKG

[0556] 20 CDR-H3.1 DYYGRFYYAMDY

[0557] 21 Peresolimab (Eh Lilly, CDR-L1.2 QASQSPNNLLA

[0558] 22 US20190270818)* CDR-L2.2 YGASDLPS

[0559] 23 CDR-L3.2 QNNYYVGPVSYA

[0560] 24 CDR-H1.2 KVSGYSLSKYDMS

[0561] 25 CDR-H2.2 IIYTSGYTDYAQKFQG

[0562] 26 CDR-H3.2 ATGNPYYTNGFNS

[0563] 27 Clone #723C2 (Boehringer CDR-L1.3 QASQSISSDYLH

[0564] 28 Ingelheim International CDR-L2.3 RTSNLET

[0565] 29 GmbH, EP4157876B1)$CDR-L3.3 QQGTSLPRA

[0566] 30 CDR-H1.3 GFTFSDYYMS

[0567] 31 CDR-H2.3 YISSGGGSSYYPDAVK

[0568] 32 CDR-H3.3 LPHYFAMDY

[0569] 33 Clone #PDlAB-6 (Pandion, CDR-L1.4 KSGQSVLYSSNQKNFLA

[0570] 34 US20240052034A1)** CDR-L2.4 WASTRES

[0571] 35 CDR-L3.4 HQYLYSWT

[0572] 36 CDR-H1.4 GFNIKDTYMH

[0573] 37 CDR-H2.4 RIDPANGDRK

[0574] 38 CDR-H3.4 SGPVYYYGSSYVMDY

[0575] 39 Anaptys Bio CDR-L1.5 RASESVDNFGISFMS

[0576] 40 CDR-L2.5 AASNPGS

[0577] 41 CDR-L3.5 QHYSSYPWT

[0578] 42 CDR-H1.5 GFTFSSYTLS

[0579] 43 CDR-H2.5 VISSGGDYAY

[0580] 44 CDR-H3.5 DHYGTSHFAY

[0581] 45 Anaptys Bio CDR-L1.6 RASESVDKYGISFMS

[0582] 46 CDR-L2.6 AASNQGS

[0583] 47 CDR-L3.6 QHYSSYPWT

[0584] 48 CDR-H1.6 GFTFSSYTMS

[0585] 49 CDR-H2.6 VISSGGNSTY

[0586]

[0587] 50 CDR-H3.6 EHYGSSHFAY

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[0590] 51 Anaptys Bio CDR-L1.7 KASQDVGTAVA

[0591] 52 CDR-L2.7 WASTRHT

[0592] 53 CDR-L3.7 QHYSSYPWT

[0593] 54 CDR-H1.7 GFTFSSYDMS

[0594] 55 CDR-H2.7 TISGGGSYTY

[0595]

[0596] 56 CDR-H3.7 PYYAMDY

[0597] *Kehry et al (2020) Pd-1 agonist and method of using same, US20220235132

[0598] #Pauliina et al (2019) Pd-1 agonist antibodies and uses thereof, US20190270818

[0599] $Blair et al (2021) Anti-pd-1 antibodies, EP4157876B1

[0600] **Higginson-Scott et al (2021) Multi-paratopic anti-pd-1 antibodies and uses thereof, US20240052034A1

[0601] Anti-TNFR Activatable Antibodies and Antigen-Binding fragments Thereof

[0602] The present disclosure provides activatable antibodies or antigen binding fragments thereof which specifically bind Tumor Necrosis Factor Receptor 2 (TNFR2), also known as tumor necrosis factor receptor superfamily member IB (TNFRSF1B) (NCBI Gene ID: 7133). Tumor necrosis factor alpha (TNFa) is a pleiotropic cytokine that mediates pro-inflammatory and anti-inflammatory functions through its two receptors: TNFR1 and TNFR2. TNFR1 is ubiquitously expressed across cell types, while TNFR2 is mainly restricted to immune cells and tumor cells. Other studies have shown that TNFR2 is involved in the phenotypic stability, proliferation, activation, and suppressive activity of Tregs (Chen et al, 2013.. / Immunol, 190(3):1076-84). Thus, TNFR2 agonism has been explored as a therapeutic approach in autoimmune disease.

[0603] Beyond its established role in immune regulation, extensive preclinical evidence demonstrates that TNFR2 signaling plays a central role in promoting immune tolerance and tissue protection across a broad range of autoimmune and inflammatory diseases. TNFR2 selectively mediates anti-inflammatory, tissue-protective, and regulatory immune functions that are distinct from the cytotoxic and pro-inflammatory signaling driven by TNFR1 (Siegmund and Wajant, Nat. Rev. Rheumatol., 2023; PMID: 37542139). These observations have positioned TNFR2 agonism as a compelling therapeutic strategy for restoring immune homeostasis in diseases where TNF blockade is insufficient, ineffective, or deleterious.

[0604] However, the development of TNFR2 agonists as a treatment for autoimmune disease has been marked by significant complexities. TNFR2 agonists are designed to selectively enhance the function of but their therapeutic utility is clouded by conflicting evidence on mechanism of action and target cell specificity. TNFR2-targeted therapies are used as agonists in autoimmune diseases to promote immune tolerance and as antagonists in cancer to disrupt immune evasion. However, existing therapeutics are delivered systemically and thus act on TNFR2 broadly as opposed to in the tissue of interest.

[0605] TNFR2 is often described in the literature as being selectively expressed on TregS. However, TNFR2 is also expressed on circulating myeloid cells and activated effector T cells. Further, TNFR2-expressing myeloid cells can deplete systemic TNFR2 agonists prior to reaching target tissues, thus 59

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[0607] reducing the effective dose available for therapeutic efficacy in diseased tissues. In addition, non-tissuespecific TNFR2 agonists can inadvertently activate effector T cells in the periphery and result in enhanced inflammation and autoimmune pathology.

[0608] The systemic limitations of current TNFR2 agonists underscore the need for innovative strategies to address challenges related to efficacy, safety, and specificity. A tissue-specific activation approach not only overcomes these hurdles but also paves the way for developing safer, more potent TNFR2 agonists tailored to specific autoimmune diseases.

[0609] In some embodiments, provided herein are anti-TNFR2 activatable monoclonal antibodies or antigen binding fragments thereof. In some embodiments, the activatable anti-TNFR2 antibody comprised of one or more binding domains that specifically bind TNFR2, one or more TNFR2 binding domain masking peptides, one or more cleavable peptides, and, optionally a linker peptide. In some embodiments, the activatable anti-TNFR2 antibody is an activatable anti-TNFR2 antibody or antigen-binding fragment thereof.

[0610] In some embodiments, the activatable anti-TNFR2 antibody or antigen binding fragment thereof is used in the treatment of an autoimmune or inflammatory disease. In some embodiments, the anti-TNFR2 activatable antibodies are used for the treatment of type 1 diabetes (T1D), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis (AD), multiple sclerosis (MS), primary biliary cholangitis, lupus nephritis, Sjorgen’s syndrome-associated dry eye, and / or autoimmune optic neuritis. TNFR2 agonism exerts potent tolerogenic and tissue-protective effects across multiple autoimmune and inflammatory disease models, including:

[0611] Graft- versus-host disease (Chopra et al., J. Exp. Med., 2016; Vargas et al., Front. Immunol., n')

[0612] Rheumatoid arthritis (Lamontain et al., Cell Mol. Immunol., 2019; Fischer et al., Arthritis Rheumatol., 2018)

[0613] Inflammatory Bowel Disease (Lubrano di Ricco et al., Eur. J. Immunol., 2020)

[0614] Multiple Sclerosis (Ronin et al., Proc. Natl. Acad. Sci. USA, 2021)

[0615] Neuroinflammation and Pain (Fischer et al., Proc. Natl. Acad. Sci. USA, 2019)

[0616] In some embodiments, the present disclosure provides methods of treating a subject with rheumatoid arthritis, comprising administering the activatable anti-TNFR2 antibody or antigen binding fragment thereof. In inflammatory arthritis models, selective TNFR2 activation has been shown to expand regulatory T cells (Tregs) and ameliorate established disease. In collagen-induced arthritis, TNFR2 stimulation increases Treg numbers and suppressive function while reducing disease severity (Lamontain et al., Cell Mol. Immunol., 2019; PMID: 29375132). Similarly, selective TNFR2 agonists induce antiinflammatory responses and alleviate experimental arthritis in vivo (Fischer et al., Arthritis Rheumatol.,

[0617] 60

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[0619] 2018; PMID: 29342501), providing strong evidence that TNFR2 signaling can counteract pathogenic effector T-cell-driven inflammation in joint disease.

[0620] In some embodiments, the present disclosure provides methods of treating a subject with inflammatory bowel disease, comprising administering the activatable anti-TNFR2 antibody or antigen binding fragment thereof. In inflammatory bowel disease models, TNFR2 signaling has been shown to enhance regulatory T-cell activation and function through NF-κB-dependent mechanisms, supporting intestinal immune tolerance (Lubrano di Ricco et al., Eur. J. Immunol., 2020; PMID: 32012260). These findings highlight TNFR2 as a disease-relevant immunoregulatory target in mucosal inflammation, where local immune balance is critical to disease control.

[0621] In some embodiments, the present disclosure provides methods of treating a subject with multiple sclerosis, comprising administering the activatable anti-TNFR2 antibody or antigen binding fragment thereof. In neuroinflammatory disease, TNFR2 signaling has emerged as a key regulator of tissue-restricted immune tolerance. In experimental autoimmune encephalomyelitis (EAE), TNFR2-expressing Tregs have been shown to exert localized control over established central nervous system autoimmunity (Ronin et al., Proc. Natl. Acad. Sci. USA, 2021; PMID: 33766913). Additional studies demonstrate that TNFR2 promotes Treg-mediated recovery in neuroinflammatory and neuropathic disease settings (Fischer et al., Proc. Natl. Acad. Sci. USA, 2019; PMID: 31391309), underscoring the importance of TNFR2 signaling in regulating inflammation within immune-privileged tissues.

[0622] Beyond classical autoimmune indications, TNFR2 agonism has also demonstrated therapeutic potential in graft-versus-host disease (GVHD), where exogenous TNFR2 activation protects against acute GVHD through host Treg expansion (Chopra et al., J. Exp. Med., 2016; PMID: 27526711), and through engineered TNFR2- specific TNF fusion proteins with enhanced in vivo activity (Vargas et al., Front. Immunol., 2022; PMID: 35769484). TNFR2 agonists have further shown tissue -protective and antiinflammatory effects in skin inflammation models (Inoue et al., J. Immunol., 2021; PMID: 33782090) and in neurodegenerative disease models, including Alzheimer’s disease (Ortí-Casañ et al., Proc. Natl. Acad. Sci. USA, 2022; PMID: 36037389).

[0623] Collectively, these studies establish TNFR2 as a highly disease-relevant immunomodulatory target across multiple autoimmune, inflammatory, and neuroinflammatory indications. However, despite this strong biological rationale, clinical translation of TNFR2 agonism has been constrained by fundamental limitations associated with systemic TNFR2 engagement. TNFR2 is not exclusively expressed on regulatory T cells but is also upregulated on activated effector T cells and myeloid cells in peripheral blood and lymphoid tissues. As a result, systemic TNFR2 agonists can be sequestered by nontarget immune populations, reducing effective delivery to diseased tissues, and may inadvertently enhance effector T-cell activation or inflammatory priming, thereby counteracting intended tolerogenic effects.

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[0626] Accordingly, while TNFR2 agonists have demonstrated potent tolerance-inducing and diseasemodifying potential in multiple preclinical models, their therapeutic utility has remained limited by the absence of technologies capable of restricting TNFR2 activation to diseased tissues. The activatable anti-TNFR antibody disclosed herein address this critical unmet need by maintaining systemic inactivity of TNFR2 agonists while enabling conditional activation selectively within inflamed tissues. By confining TNFR2 signaling to sites enriched for pathogenic immune activity, the present disclosure enables safer, more potent, and more durable TNFR2-based immunotherapies while mitigating the systemic liabilities associated with broad TNFR2 engagement.

[0627] In some embodiments, the activatable anti-TNFR2 antibody is specific to human, murine, or primate TNFR2. In some embodiments, provided herein are exemplary anti-TNFR2 activatable antibodies or antigen binding fragments thereof as set forth in Table 4A and Table 4B. In some embodiments, provided herein are anti-TNFR2 activatable antibodies or antigen binding fragments thereof, comprising six CDR sequences set forth in Table 4A and Table 4B.

[0628] In some embodiments, the anti-TNFR2 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-TNFR2 activatable antibody comprises 6 CDRs.

[0629] In some embodiments, the anti-TNFR2 activatable antibody or antigen binding fragment thereof comprises six CDR sequences set forth in SEQ ID NOs: 57-171 (Wajant et al (2022), Novel tnfr2 binding molecules, WO2022 / 207921 A9, incorporated herein by reference).

[0630] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 84, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 85; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 57, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 83, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 86; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 58, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71.

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[0633] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 83, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 87; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72.

[0634] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 83, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 88; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 60, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 73.

[0635] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 84, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 89; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 61, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 74.

[0636] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 83, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 90; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 62, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 75.

[0637] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 82, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 83, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 91; and / or wherein the heavy chain variable region comprises (1) a

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[0640] CDR-H1 comprising the amino acid sequence of SEQ ID NO: 63, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70.

[0641] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 82, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 84, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 92; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 64, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 76.

[0642] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 82, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 84, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 93; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 64, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77.

[0643] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 82, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 83, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 94; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 58, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 78.

[0644] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 83, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 95; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 79.

[0645] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 83, (3) a CDR-L3 comprising the 64

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[0647] amino acid sequence of SEQ ID NO: 95; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 65, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 79.

[0648] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 83, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 92; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 66, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 79.

[0649] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 82, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 84, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 96; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 64, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 77.

[0650] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 83, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 97; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 66, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 80.

[0651] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 84, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 85; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 57, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 67, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 70.

[0652] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 101,

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[0655] (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 102, (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 103; and / or wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 98, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 99, (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 100.

[0656] In some embodiments, the anti-TNFR activatable antibody or antigen binding fragment thereof is comprised of two sets of three variable heavy domain of heavy chain (VHH) antibodies forming a hexameric TNFR2 agonist. In some embodiments, the anti-TNFR activatable antibody comprises the amino acid sequences set forth in SEQ ID NO: 104 or 105.

[0657] In some embodiments, the activatable anti-TNFR2 antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 101, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 102, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 103.

[0658] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 106, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 111, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 124.

[0659] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 106, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 112, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 124.

[0660] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 106, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

[0661] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 106, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 124.

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[0664] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 106, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 116, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 126.

[0665] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 106, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 111, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 127.

[0666] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 106, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 111, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 128.

[0667] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 106, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 111, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 124.

[0668] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 106, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 111, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 129.

[0669] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 106, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 129.

[0670] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (2) a CDR-L2 comprising the amino

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[0673] acid sequence of SEQ ID NO: 116, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 120.

[0674] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 131.

[0675] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 119, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 131.

[0676] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 120, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 131.

[0677] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 120, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 130.

[0678] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 130.

[0679] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 121, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 131.

[0680] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a 68

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[0682] CDR-L1 comprising the amino acid sequence of SEQ ID NO: 108, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 122, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 132.

[0683] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 108, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 122, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0684] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a light chain variable region, wherein the light chain variable region comprises (1) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 110, (2) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (3) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 134.

[0685] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 110, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 123, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134.

[0686] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 154.

[0687] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 136, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 155.

[0688] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 156.

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[0691] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 157.

[0692] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 137, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 157.

[0693] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 138, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 158.

[0694] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 139, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 159.

[0695] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 140, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 160.

[0696] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 141, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 161.

[0697] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, (2) a CDR-H2 comprising the amino

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[0700] acid sequence of SEQ ID NO: 149, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 161.

[0701] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 162.

[0702] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 142, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 163.

[0703] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 141, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 164.

[0704] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 141, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 154.

[0705] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 141, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 150, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 165.

[0706] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 166.

[0707] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1)

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[0710] a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 144, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 166.

[0711] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 145, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 167.

[0712] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 152, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 166.

[0713] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 146, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 166.

[0714] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 168.

[0715] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 143, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 151, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 167.

[0716] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 147, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 153, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 169.

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[0718] FH13277368.1Attorney Docket No: TGY-00125

[0719] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 147, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 153, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 170.

[0720] In some embodiments, the activatable anti-TNFR antibody or antigen binding fragment of the disclosure comprises a heavy chain variable region, wherein the heavy chain variable region comprises (1) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 147, (2) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 153, and (3) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 171.

[0721] In some embodiments, the activatable anti-TNFR2 antibody comprises any combination of the heavy chain CDR sequences set forth in SEQ ID NOs: 135-171 with any of the light chain CDR sequences set forth in SEQ ID NOs: 106-134.

[0722] It is well known in the art that mutations may be introduced into proteins having a well characterized sequence and three dimensional structure such as an antibody without the loss of function. Accordingly, in some embodiments, the activatable antibody comprises a variant of the light chain and heavy chain CDRs as set forth in SEQ ID NOs: 57-171. The variant may differ in one or more amino acids as compared to the framework regions i.e. FR1, FR2, FR3 and FR4 of the parent VH and / or VL sequence, e.g., in one or more framework regions, but still allows the antigen-binding region to retain at least a substantial proportion (at least about 90 percent, 95 percent or more) or even retain all of the affinity and / or specificity of the parent antibody. Typically, such functional variants retain significant sequence identity to the parent sequence. Exemplary variants include those which differ from the respective parent VH or VL region by 5 or less, such as 5, 4, 3, 2 or 1 mutation(s) such as substitutions, of amino acid residues. Exemplary variants include those which differ from the VH and / or VL regions of the parent sequences mainly by conservative amino acid substitutions; for instance, 5, such as 5, 4, 3, 2 or 1 of the amino acid substitutions in the variant can be conservative. In a further aspect of the invention the antibody may comprise at most 1, 2 or 3 mutations in the VH framework regions and / or in the VL framework regions, respectively. Such mutations may be substitutions. It is preferred that such substitutions do not significantly change the binding affinity and / or binding specificity of the acti vatable antibodies of the invention.

[0723] In some embodiments, the anti-TNFR2 activatable antibody or antigen binding fragment thereof comprises one or more binding domains. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-L1 comprising at least 80% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 81, 82, 101, 106, 107, 108, 109, and 110. In some embodiments, the anti-TNFR2

[0724] 73

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[0726] activatable antibody comprises a CDR-L1 comprising at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 81, 82, 101, 106, 107, 108, 109, and 110. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-L1 comprising at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 81, 82, 101, 106, 107, 108, 109, and 110. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-L1 comprising at least 95% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 81, 82, 101, 106, 107, 108, 109, 110.

[0727] In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-L2 comprising at least 80% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 83-84, 102, 111-117, and 119-123. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-L2 comprising at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 83-84, 102, 111-117, and 119-123. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-L2 comprising at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 83-84, 102, 111-117, and 119-123. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-L2 comprising at least 95% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 83-84, 102, 111-117, and 119-123.

[0728] In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-L3 comprising at least 80% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 85- 97, 103, 120, and 124-134. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-L3 comprising at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 85- 97, 103, 120, and 124-134. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-L3 comprising at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 85- 97, 103, 120, and 124-134. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-L3 comprising at least 95% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 85- 97, 103, 120, and 124-134.

[0729] In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H1 comprising at least 80% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 57-66, 98, and 135-147. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H1 comprising at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 57-66, 98, and 135-147. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H1 comprising at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 57-66, 98, and 135-147. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H1 comprising at least 95% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 57-66, 98, and 135-147.

[0730] 74

[0731] FH13277368.1Attorney Docket No: TGY-00125

[0732] In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H2 comprising at least 80% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 67- 69, 99, and 148-153. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H2 comprising at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 67- 69, 99, and 148-153. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H2 comprising at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 67- 69, 99, and 148-153. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H2 comprising at least 95% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 67- 69, 99, and 148-153.

[0733] In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H3 comprising at least 80% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 70- 80, 100, and 154-171. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H3 comprising at least 85% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 70- 80, 100, and 154-171. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H3 comprising at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 70- 80, 100, and 154-171. In some embodiments, the anti-TNFR2 activatable antibody comprises a CDR-H3 comprising at least 95% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 70- 80, 100, and 154-171.

[0734] In some embodiments, the anti-TNFR2 activatable antibody or antigen binding fragment thereof comprises any one of the following sequences: HFB200301 (Wei et al (2021) Anti-tnfr2 antibody and uses thereof, US20230045791A1, incorporated herein by reference); BI-1910 and / or BI-1808 (Frendeus et al (2019) Novel agonistic antibody molecules, W02020089473A2, incorporated herein by reference), DT-001 (Dualyx, Wuerzburg University), TRB-061 (TRexBio), and NKTR-065 (Inbar et al (2021) Antitumor necrosis factor ( tnfr2) antibodies and uses thereof W02022003690A2, incorporated herein by reference). In some embodiments, the anti-TNFR2 activatable antibody or antigen binding fragment thereof comprises NKTR-0165.

[0735] TABLE 4A: Exemplary Anti-TNFR2 Activatable Antibodies

[0736] Description Antibody CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3 Biologic Anti- GFNIKA IDPDNGD SREGQDG QDVSTA PAS QQPLTTPPT Design Ltd TNFR2 #1 YY (SEQ T (SEQ ID NYAWQDL (SEQ ID (SEQ ID (SEQ ID NO: (W0202200 ID NO: NO: 67) (SEQ ID NO: 81) NO: 84) 85) 3690A2)* 57) NO: 70)

[0737] Anti- GFNIRA IDPTNGD TREGQDG QDVSTA SAS QQHYTPYP TNFR2 #2 MY(SEQ T (SEQ ID NYAFDK (SEQ ID (SEQ ID T (SEQ ID ID NO: NO: 68) (SEQ ID NO: 81) NO: 83) NO: 86)

[0738]

[0739] 58) NO: 71)

[0740] 75

[0741] FH13277368.1Attorney Docket No: TGY-00125

[0742] Anti- GFNIRD IDPTNGD SREMADG QDVSTA SAS QQPNYTTPP TNFR2 #3 TY (SEQ T (SEQ ID YNAFDL (SEQ ID (SEQ ID T (SEQ ID ID NO: NO: 68) (SEQ ID NO: 81) NO: 83) NO: 87) 59) NO: 72)

[0743] Anti- GFNIRAT ISPDDNG SRDLADG QDVSTA SAS QQHGYYTP TNFR2 #4 Y (SEQ DT (SEQ YNAFDL (SEQ ID (SEQ ID PT (SEQ ID ID NO: ID NO: 69) (SEQ ID NO: 81) NO: 83) NO: 88) 60) NO: 73)

[0744] Anti- GFNIRDI IDPDNGD TREGADG QDVSTA PAS QQPYTPYPT TNFR2 #5 TY (SEQ T (SEQ ID YNAFDL (SEQ ID (SEQ ID (SEQ ID NO:

[0745] ID NO: NO: 67) (SEQ ID NO: 81) NO: 84) 89) 61) NO: 74)

[0746] Anti- GFNIRAF IDPTNGD TREGEDG QDVSTA SAS QQPRYYTPP TNFR2 #6 Y (SEQ T (SEQ ID YNAFDQ (SEQ ID (SEQ ID T (SEQ ID ID NO: NO: 68) (SEQ ID NO: 81) NO: 83) NO: 90) 62) NO: 75)

[0747] Anti- GFNIKD IDPTNGD SREGQDG QDISTA SAS QQPSLTTPP TNFR2 #7 FDF (SEQ T (SEQ ID NYAWQDL (SEQ ID (SEQ ID T (SEQ ID ID NO: NO: 68) (SEQ ID NO: 82) NO: 83) NO: 91) 63) NO: 70)

[0748] Anti- GFNIKD IDPTNGD SREGQDG QDISTA PAS QQHPYTPPT TNFR2 #8 YF (SEQ T (SEQ ID NYAWDDQ (SEQ ID (SEQ ID (SEQ ID NO:

[0749] ID NO: NO: 68) (SEQ ID NO: 82) NO: 84) 92) 64) NO: 76)

[0750] Anti- GFNIKD IDPTNGD SREGQDG QDISTA PAS QQHGYTPP TNFR2 #9 YF (SEQ T (SEQ ID NYAWDM (SEQ ID (SEQ ID T (SEQ ID ID NO: NO: 68) (SEQ ID NO: 82) NO: 84) NO: 93) 64) NO: 77)

[0751] Anti- GFNIRA IDPTNGD TREGQDG QDISTA SAS QQPNLTTPP TNFR2 MY (SEQ T (SEQ ID NYAFDM (SEQ ID (SEQ ID T (SEQ ID #10 ID NO: NO: 68) (SEQ ID NO: 82) NO: 83) NO: 94)

[0752] 58) NO: 78)

[0753] Anti- GFNIRA IDPTNGD SREGQDG QDVSTA SAS QQPGYTTPP TNFR2 YY (SEQ T (SEQ ID NYAWDL (SEQ ID (SEQ ID T (SEQ ID #11 ID NO: NO: 68) (SEQ ID NO: 81) NO: 83) NO: 95)

[0754] 65) NO: 79)

[0755] Anti- GFNIRD IDPTNGD SREGQDG QDVSTA SAS QQHPYTPPT TNFR2 YF (SEQ T (SEQ ID NYAWDL (SEQ ID (SEQ ID (SEQ ID NO: #12 ID NO: NO: 68) (SEQ ID NO: 81) NO: 83) 92)

[0756] 66) NO: 79)

[0757] Anti- GFNIKD IDPTNGD SREGQDG QDISTA PAS QQPLYTPPT TNFR2 YF (SEQ T (SEQ ID NYAWDM (SEQ ID (SEQ ID (SEQ ID NO: #13 ID NO: NO: 68) (SEQ ID NO: 82) NO: 84) 96)

[0758] 64) NO: 77)

[0759] Anti- GFNIRD ISPDDNG TREGEDG QDVSTA SAS QQHAYTPP TNFR2 YF (SEQ DT (SEQ NYAFDM (SEQ ID (SEQ ID T (SEQ ID #14 ID NO: ID NO: 69) (SEQ ID NO: 81) NO: 83) NO: 97)

[0760] 66) NO: 80)

[0761] Julius- Anti- SYDIN WIYPRDG LTGPYYW KASQDV WASTR QQYYSVPP Maximilian TNFR2 (SEQ ID DTKYNE FDV (SEQ DTAVA HT (SEQ T (SEQ ID s- #15 NO: 98) KFKGKAI ID NO: 100) (SEQ ID ID NO: NO: 103) Universitat (SEQ ID NO: 101) 102)

[0762] Wurzburg NO: 99)

[0763] (WO2022 / 2

[0764]

[0765] 07921A9)#

[0766] 76

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[0768] TABLE 4B: Exemplary Anti-TNFR2 Activatable Antibodies

[0769] SEQ Antibody Sequence

[0770] ID NO

[0771] 104 3XVHHC188(G4 DVQLVESGGGSVQTGGSLTLSCAISGSTSERYCLGWFRQAPGREREGVAAT S)l-Fc(Durv)# SLTGRGAQFYADSVKGRFTISLDDAKNTLYLQMDSLRPDDTAVYYCAEDV GFLCGYDSDNDPFYDWGQGTQVTVSSGGGGSDVQLVESGGGSVQTGGSL

[0772] Linker TLSCAISGSTSERYCLGWFRQAPGREREGVAATSLTGRGAQFYADSVKGRF Hinge TISLDDAKNTLYLQMDSLRPDDTAVYYCAEDVGFLCGYDSDNDPFYDWG Fc (Durvalumab) QGTQVTVSSGGGGSDVQLVESGGGSVQTGGSLTLSCAISGSTSERYCLGWF RQAPGREREGVAATSLTGRGAQFYADSVKGRFTISLDDAKNTLYLQMDSLR PDDTAVYYCAEDVGFLCGYDSDNDPFYDWGOGTQVTVSSEPKSDKTHTC PPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPASIEKTISKAKGOPREPQVYTLPPSREEMTKNOVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSV MHEALHNHYTOKSLSLSPGK

[0773] 105 3XVHHC188- EVQLVESGGGLVQPGGSLRLSCAISGSTSERYCLGWFRQAPGKGREGVAAT varl4(G4S)l- SLTGRGAQFYADSVKGRFTISLDDAKNTLYLQMNSLRAEDTAVYYCAEDV Fc(Durv)#GFLCGYDTDNEPFYDWGQGTLVTVSSCGCGSEVQLVESGGGLVQPGGSLR LSCAISGSTSERYCLGWFRQAPGKGREGVAATSLTGRGAQFYADSVKGRFTI

[0774] Linker SLDDAKNTLYLQMNSLRAEDTAVYYCAEDVGFLCGYDTDNEPFYDWGQG Hinge TLVTVSSCGCGSEVQLVESGGGLVQPGGSLRLSCAISGSTSERYCLGWFRQ Fc (Durvalumab) APGKGREGVAATSLTGRGAQFYADSVKGRFTISLDDAKNTLYLQMNSLRAE DTAVYYCAEDVGFLCGYDTDNEPFYDWGOGTLVTVSSEPKSDKTHTCPP CPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKAL PASIEKTISKAKGOPREPQVYTLPPSREEMTKNOVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHE

[0775]

[0776] ALHNHYTQKSLSLSPGK

[0777] *Inbar et al (2021) Anti-tumornecrosis factor receptor (tnfr2) antibodies and uses thereof W02022003690A2#Wajant et al (2022), Novel tnfr2 binding molecules, WO2022 / 207921

[0778] Table 5: Anti-TNFR2 Light Chain CDR Sequences- W02022003690A2*

[0779] CDR-L1 SEQ ID CDR-L2 SEQ ID CDR-L3 SEQ ID NO NO NO GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 124

[0780] YDDYGFLKH GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 124

[0781] YDDYGFLKH GDTFTSYS 106 IITDLDDT 112 AGVYEGPNSEGS 124

[0782] YDDYGFLKH GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 124

[0783] YDDYGFLKH GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 124

[0784] YDDYGFLKH GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 124

[0785] YDDYGFLKH GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 124

[0786]

[0787] YDDYGFLKH

[0788] 77

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[0790] GDTFTSYS 106 IITIPDDT 113 AGVYEGPNSEGS 124

[0791] YDDYGFLKH GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 124

[0792] YDDYGFLKH GDTFTSYS 106 IITIDDDT 114 AGVYEGPNSEGS 125

[0793] YDDYGFYKH GDTFTSYS 106 IITILDDS 115 AGVYEGPNSEGS 124

[0794] YDDYGFLKH GDTFTSYS 106 IITIDDDT 114 AGVYEGPNSEGS 125

[0795] YDDYGFYKH GDTFTSYS 106 IITIYDDT 116 AGVYEGPNSEGS 126

[0796] YDDYGFHK GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 127

[0797] YDDYGFLKY GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 128

[0798] YDDYGALKH GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 124

[0799] YDDYGFLKH GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 129

[0800] YDDYGQLKH GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 129

[0801] YDDYGQLKH GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 124

[0802] YDDYGFLKH GDTFTSYS 106 IITILDDT 111 AGVYEGPNSEGS 124

[0803] YDDYGFLKH GDTFTSYS 106 IITIDDDT 114 AGVYEGPNSEGS 129

[0804] YDDYGQLKH GDTFTSYS 106 IITIDDDT 114 AGVYEGPNSEGS 129

[0805] YDDYGQLKH GYTFTDLG 107 INTHTGMP 116 ARDHPLGLDY 120 GYTFTDLG 107 INTHTGEP 117 TRDHPLGLDY 131 GYTFTDLG 107 INTHTGEP 117 TRDHPLGLDY 131 GYTFTDLG 107 INTHTGKP 119 TRDHPLGLDY 131 GYTFTDLG 107 INTQTGEP 120 TRDHPLGLDY 131 GYTFTDLG 107 INTQTGEP 120 ARDHPLGLDY 130 GYTFTDLG 107 INTHTGEP 117 TRDHPLGLDY 131 GYTFTDLG 107 INTHTGEP 117 ARDHPLGLDY 130 GYTFTDLG 107 INTHTGEP 117 TRDHPLGLDY 131 GYTFTDLG 107 INTHTGEP 117 TRDHPLGLDY 131

[0806]

[0807] 78

[0808] FH13277368.1Attorney Docket No: TGY-00125

[0809] GYTFTDLG 107 INTITGEP 121 TRDHPLGLDY 131 GYTFTDLG 107 INTHTGEP 117 TRDHPLGLDY 131 GFINIRDTY 108 IDPDNGDT 122 SRDLADGYNAF 132

[0810] DR GFINIRDTF 109 IDPDNGDT 122 SREGEDGNYAW 133

[0811] DL GFINIRATY 110 IDPTNGDT 123 TREGDDGNYAW 134

[0812]

[0813] DK

[0814] *Inbar et al (2021) Anti-tumornecrosis factor receptor (tnfr2) antibodies and uses thereof W02022003690A2

[0815] Table 6: Anti-TNFR2 Heavy Chain CDR Sequences- W02022003690A2*

[0816] CDR-H1 SEQ ID CDR-H2 SEQ ID CDR-H3 SEQ ID NO NO NO ESVAND 135 GAS 148 QQYANWPPRET 154 ESVAND 135 GAS 148 QQYANWPPRET 154 ESGAND 136 GAS 148 QQYANWPRRGT 155 ESVAND 135 GAS 148 QQYYNWPWRET 156 ESVAND 135 GAS 148 QQYAIWPWRET 157 RSVAND 137 GAS 148 QQYAIWPWRET 157 ESGANN 138 GAS 148 QQYANWPRET 158 ESVATD 139 GAS 148 QQYDNWPWRET 159 ESVRND 140 GAS 148 QQYANWPHPRET 160 ESVGND 141 GAS 148 QQYANWPWRET 161 ESVAND 135 DAS 149 QQYANWPWRET 161 ESVAND 135 GAS 148 QQGANWPWPRET 162 ESVAND 135 GAS 148 QQYANWPPRET 154 ESVAVD 142 GAS 148 QQYANPKPRET 163 ESVAND 135 GAS 148 QQYANWPPRET 154 ESGAND 136 GAS 148 QQYANWPRRGT 155 ESVAND 135 GAS 148 QQYANWPPRET 154 ESVAND 135 GAS 148 QQYANWPPRET 154 ESVAND 135 GAS 148 QQYANWPPRET 154 ESVGND 141 GAS 148 QQYANVPPRET 164 ESVGND 141 GAS 148 QQYANWPPRET 154 ESVGND 141 GIS 150 QQYANDPPRET 165 QSQVDVNGVSY 143 KAS 151 QQSREDPYT 166 QSQVDVNGVSY 143 KAS 151 QQSREDPYT 166 QSQVDNGLSY 144 KAS 151 QQSREDPYT 166 QSQVDVNGVSY 143 KAS 151 QQSREDPYT 166 QSQVDVAGVSY 145 KAS 151 QQAREDYPYT 167 QSQVDVNGVSY 143 KAS 151 QQSREDPYT 166

[0817]

[0818] 79

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[0820] QSQVDVNGVSY 143 KAS 151 QQSREDPYT 166 QSQVDVNGVSY 143 KAV 152 QQSREDPYT 166 QSLDVNGVSY 146 KAS 151 QQSREDPYT 166 QSQVDVNGVSY 143 KAS 151 QQNREDPYT 168 QSQVDVNGVSY 143 KAS 151 QQSREDPYT 166 QSQVDVNGVSY 143 KAS 151 QQAREDYPYT 167 QDVSTA 147 SAS 153 QQPYTPYPT 169 QDVSTA 147 SAS 153 QQPYTPPT 170 QDVSTA 147 SAS 153 QQPSYTPPT 171

[0821]

[0822] *Inbar et al (2021) Anti-tumornecrosis factor receptor (tnfr2) antibodies and uses thereof W02022003690A2

[0823] Anti-CD2 Activatable Antibodies and Antigen-Binding fragments Thereof

[0824] The principles of tissue-restricted activation by targeting T cell antigens are not limited to CD3. Rather, the same rationale and technological advantages apply broadly to other T-cell-associated targets whose systemic modulation is constrained by dose-limiting toxicity, lack of tissue selectivity, or insufficient durability. In some embodiments, activatable antibodies of the present disclosure may be directed against additional T-cell surface molecules or signaling components, including CD2, the T-cell receptor (TCR) complex, CD4, CD8, or other T-cell-associated antigens involved in T-cell activation, survival, regulatory or effector function. As with CD3, these targets are broadly expressed on circulating T cells but exert disease-driving activity within localized inflammatory microenvironments. Accordingly, conditional, tissue-restricted activation of antibodies directed to such targets enables selective modulation or elimination of pathogenic T cells within diseased tissues while minimizing systemic immune perturbation, thereby extending the therapeutic utility of the disclosed activatable antibody platforms across a wide range of autoimmune and inflammatory indications

[0825] In some embodiments, the disclosure provides activatable anti-CD2 antibodies and antigen binding fragments thereof. CD2 is a cell adhesion molecule found on the surface of T cells and natural killer (NK) cells, where it can also function as a co- stimulatory receptor. Anti-CD2 antibodies have been shown to have immunomodulatory effects through the depletion of CD2+ cells. CD2 expression is increased on memory T cells relative to naive T cells and on activated T cells relative to resting T cells (Lo et al, 2011. American J of Transplantation, 11(1), 22-33). Thus, anti-CD2 antibodies can selectively target the alloreactive memory T cells associated with autoimmunity. In some embodiments, provided herein are anti-CD2 activatable monoclonal antibodies or antigen binding fragments thereof. The present disclosure provides activatable anti-CD2 antibodies comprised of one or more binding domains that specifically bind CD2, one or more CD2 binding domain masking peptides, one or more cleavable peptides, and, optionally a linker peptide. In some embodiments, the activatable anti-CD2 antibody is an activatable anti-CD2 antibody or antigen-binding fragment thereof.

[0826] 80

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[0828] In some embodiments, an activatable anti-CD2 antibody or antigen binding fragment thereof is used in the treatment of an autoimmune or inflammatory disease. In some embodiments, anti-CD2 activatable antibodies are used for the treatment of type 1 diabetes (T1D), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), atopic dermatitis (AD), multiple sclerosis (MS), primary biliary cholangitis, lupus nephritis, Sjorgen’s syndrome-associated dry eye, and / or autoimmune optic neuritis.

[0829] In some embodiments, an activatable anti-CD2 antibody or antigen binding fragment thereof is specific to human, murine, or primate CD2. In some embodiments, provided herein are anti-CD2 activatable antibodies comprising the variable regions of Alefacept (Amevive), comprising: (1) the heavy chain variable region set forth in SEQ ID NO: 172 and (2) the light chain variable region set forth in SEQ ID NO: 173 (Vaishnaw et al (2002) Methods for treating or preventing skin disorders using cd2-binding agents, US20040170635A1, incorporated herein by reference).

[0830] It is well known in the art that mutations may be introduced into proteins having a well characterized sequence and three dimensional structure such as an antibody without the loss of function. Accordingly, in some embodiments, the activatable antibody comprises a variant of the VH or VL set forth by SEQ ID NOs: 172 or 173, respectively. The variant may differ in one or more amino acids as compared to the framework regions i.e. FR1, FR2, FR3 and FR4 of the parent VH and / or VL sequence, e.g., in one or more framework regions, but still allows the antigen-binding region to retain at least a substantial proportion (at least about 90 percent, 95 percent or more) or even retain all of the affinity and / or specificity of the parent antibody. Typically, such functional variants retain significant sequence identity to the parent sequence. Exemplary variants include those which differ from the respective parent VH or VL region by 5 or less, such as 5, 4, 3, 2 or 1 mutation(s) such as substitutions, of amino acid residues. Exemplary variants include those which differ from the VH and / or VL regions of the parent sequences mainly by conservative amino acid substitutions; for instance, 5, such as 5, 4, 3, 2 or 1 of the amino acid substitutions in the variant can be conservative. In a further aspect of the invention the antibody may comprise at most 1, 2 or 3 mutations in the VH framework regions and / or in the VL framework regions, respectively. Such mutations may be substitutions. It is preferred that such substitutions do not significantly change the binding affinity and / or binding specificity of the acti vatable antibodies of the invention.

[0831] In some embodiments, the activatable anti-CD2 antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 172. In some embodiments, the heavy chain variable region comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 172. In some embodiments, the heavy chain variable region comprises at least 90% sequence identity to the amino acid

[0832] 81

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[0834] sequence set forth in SEQ ID NO: 172. In some embodiments, the heavy chain variable region comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 172.

[0835] In some embodiments, the activatable anti-CD2 antibody or antigen binding fragment thereof comprises a light chain variable region comprising at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 173. In some embodiments, the light chain variable region comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 173. In some embodiments, the light chain variable region comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 173. In some embodiments, the light chain variable region comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 173. In some embodiments, the anti-CD2 variable heavy chain and variable light chain are provided in Table 7.

[0836] TABLE 7: Anti-CD2 Variable Chain Sequences of Alefacept (Amevive)*

[0837] SEQ ID Description Sequence

[0838] NO

[0839] 172 VH FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEF RAFSSFKNRVYLDTVSGSLTIYNLTSSDEDEYEMESPNITDTMKF FLYVDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKE

[0840] 173 VL YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ

[0841] VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

[0842]

[0843] SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Vaishnaw et al (2002) Methods for treating or preventing skin disorders using cd2-binding agents, US20040170635A1.

[0844] Masking Peptide

[0845] A binding domain masking peptide or “masking peptide” as provided herein refers to a peptide which binds to or interacts with one or more antigen binding domains (one or more CDRs) of the activatable antibody of the disclosure and inhibits binding to an immunomodulatory T cell molecule. When bound to the antigen binding domain, the masking peptide blocks, inhibits, or otherwise prevents the activity of the binding domain. In some embodiments, the masking peptide comprises an anti -idiotype antibody or fragment thereof which binds to the acti vatable antibody or fragment thereof to inhibit binding to, an immunomodulatory T cell molecule. In some embodiments, the masking peptide is comprised of six peptides each binding to the six CDRs of the activatable antibody to inhibit binding of the light chain variable region and the heavy chain variable region to an immunomodulatory T cell molecule.

[0846] In certain embodiments, the masking peptide is designed using structure-guided or computational approaches informed by three-dimensional structural information of the antibody-antigen complex, such 82

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[0848] that the masking peptide sterically interferes with antigen binding when linked proximal to the antigenbinding domain. Such design may include in silico modeling, empirical screening, or combinations thereof.

[0849] In some embodiments, the masking peptide does not comprise an anti-idiotype antibody. In some embodiments, the masking peptide reduces target engagement through steric hindrance, spatial occlusion, competitive epitope adjacency, conformational restriction, or a combination thereof. In some embodiments, the masking peptide does not bind to the idiotype, paratope, or CDRs of the antibody antigen binding site. In some embodiments, the masking peptide comprises two identical masking peptides. In some embodiments, the masking peptide comprises two leucine zipper coiled-coils. In some embodiments, the two identical masking peptides form parallel heterodimeric coiled-coils with high intercoil affinities to reversibly impair antibody binding and biological function.

[0850] In some embodiments, the masking peptide comprises the sequences set forth in Table 7. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NOs: 174-191. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 174. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 175. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 176. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 177. In some embo204diments, the masking peptide comprises the sequence set forth in SEQ ID NO: 178. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 179. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 180. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 181. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 182. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 183. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 184. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 185. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 186. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 187. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 188. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 189. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 190. In some embodiments, the masking peptide comprises the sequence set forth in SEQ ID NO: 191.

[0851] In some embodiments, the masking peptide comprises three peptides comprising any of the sequences set forth in SEQ ID NO: 174-191. In some embodiments, the masking peptide comprises three peptides comprising the sequences set forth in SEQ ID NO: 174-176. In some embodiments, the masking 83

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[0853] peptide comprises three peptides comprising the sequences set forth in SEQ ID NO: 177-179. In some embodiments, the masking peptide comprises three peptides comprising the sequences set forth in SEQ ID NO: 180-182. In some embodiments, the masking peptide comprises three peptides comprising the sequences set forth in SEQ ID NO: 183-185. In some embodiments, the masking peptide comprises three peptides comprising the sequences set forth in SEQ ID NO: 186-188.

[0854] In some embodiments, the masking peptide comprises 6 peptides comprising the sequences set forth in SEQ ID NO: 174-179. In some embodiments, the masking peptide comprises 6 peptides comprising the sequences set forth in SEQ ID NO: 180-185. In some embodiments, the masking peptide comprises 6 peptides comprising the sequences set forth in SEQ ID NO: 186-191.

[0855] In some embodiments, the masking peptide comprises an amino acid sequence set forth in SEQ ID NO: 445-447 or 449. In some embodiments, the masking peptide comprises an amino acid sequence set forth in SEQ ID NO: 445. In some embodiments, the masking peptide comprises an amino acid sequence set forth in SEQ ID NO: 446. In some embodiments, the masking peptide comprises an amino acid sequence set forth in SEQ ID NO: 447. In some embodiments, the masking peptide comprises an amino acid sequence set forth in SEQ ID NO: 449.

[0856] In some embodiments, the masking peptide set forth in SEQ ID NO: 447 may be used to mask any antibody. In some embodiments, the masking peptide set forth in SEQ ID NO: 447 reduces target engagement through steric hindrance, spatial occlusion, competitive epitope adjacency, conformational restriction, or a combination thereof. In some embodiments, the activatable antibody comprises two identical masking peptides set forth in SEQ ID NO: 447. In some embodiments, the masking peptide set forth in SEQ ID NO: 447 comprises two leucine zipper coiled-coils. In some embodiments, the two identical masking peptides set forth in SEQ ID NO: 447 form parallel heterodimeric coiled-coils with high inter-coil affinities to reversibly impair antibody binding and biological function.

[0857] In some embodiments, the masking peptide has a length of at least 4 amino acids. In some embodiments, the masking peptide has a length of 4 amino acids. In some embodiments, the masking peptide has a length of 5 amino acids. In some embodiments, the masking peptide has a length of 6 amino acids. In some embodiments, the masking peptide has a length of 7 amino acids. In some embodiments, the masking peptide has a length of 8 amino acids. In some embodiments, the masking peptide has a length of 9 amino acids. In some embodiments, the masking peptide has a length of 10 amino acids. In some embodiments, the masking peptide has a length of 11 amino acids. In some embodiments, the masking peptide has a length of 12 amino acids. In some embodiments, the masking peptide has a length of 13 amino acids. In some embodiments, the masking peptide has a length of 14 amino acids. In some embodiments, the masking peptide has a length of 15 amino acids. In some embodiments, the masking peptide has a length of 16 amino acids. In some embodiments, the masking peptide has a length of 17

[0858] 84

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[0860] amino acids. In some embodiments, the masking peptide has a length of 18 amino acids. In some embodiments, the masking peptide has a length of 19 amino acids. In some embodiments, the masking peptide has a length of 20 amino acids.

[0861] In some embodiments, the masking peptide is linked to the N-terminus of the light chain or the heavy chain of the activatable antibody or antigen binding fragment thereof. In some embodiments, the masking peptide is linked to the N-terminus of the light chain variable region or the heavy chain variable region of the activatable antibody or antigen binding fragment thereof. In some embodiments, the masking peptide is linked to the C-terminus of the light chain or heavy chain of the activatable antibody or antigen binding fragment thereof. In some embodiments, the masking peptide is linked to the C-terminus of the light chain variable region or heavy chain variable region of the activatable antibody or antigen binding fragment thereof.

[0862] In some embodiments, the masking peptide comprises a first masking peptide and a second masking peptide. In some embodiments, the first masking peptide is linked to the VH domain and a second masking peptide is linked to the VL domain. In some embodiments, the first masking peptide is linked to the VH domain via a protease-cleavable linker. In some embodiments, the second masking peptide is linked to the VL domain via a protease-cleavable linker.

[0863] In some embodiments, the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VH domain. In some embodiments, the masking peptide is linked directly to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VH domain. In some embodiments, the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked directly to the VH domain.

[0864] In some embodiments, the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VL domain. In some embodiments, the masking peptide is linked directly to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VL domain. In some embodiments, the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked directly to the VL domain. It is well known in the art that mutations may be introduced into proteins having a well characterized sequence and three dimensional structure such as a peptide without the loss of function. Accordingly, in some embodiments, the masking peptide comprises a variant of the amino acid sequences set forth in SEQ ID NOs: 174-191, 445-447 or 449. The variant may differ in one or more amino acids as compared to SEQ ID NOs: 174-191, 445-447 or 449 retains the affinity and / or specificity of the parent masking peptide. Typically, such functional variants retain significant sequence identity to the parent sequence. Exemplary variants include those which differ from the respective parent peptide by 5 or less, such as 5, 4, 3, 2 or 1 mutation(s) such as substitutions, of amino acid residues. Exemplary variants include those 85

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[0866] which differ from the parent sequences mainly by conservative amino acid substitutions; for instance, 5, such as 5, 4, 3, 2 or 1 of the amino acid substitutions in the variant can be conservative. It is preferred that such substitutions do not significantly change the binding affinity and / or binding specificity of the masking peptide.

[0867] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 174. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 174. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 174. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 174. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 174.

[0868] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 175. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 175. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 175. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 175. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 175.

[0869] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 176. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 176. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 176. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 176. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 176.

[0870] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 177. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 177. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 177. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 177. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 177.

[0871] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 178. In some embodiments, a masking peptide comprises at least 86

[0872] FH13277368.1Attorney Docket No: TGY-00125

[0873] 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 178. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 178. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 178. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 178.

[0874] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 179. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 179. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 179. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 179. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 179.

[0875] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 180. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 180. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 180. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 180. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 180.

[0876] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 181. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 181. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 181. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 181. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 181.

[0877] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 182. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 182. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 182. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 182. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 182.

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[0880] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 183. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 183. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 183. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 183. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 183.

[0881] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 184. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 184. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 184. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 184. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 184.

[0882] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 185. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 185. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 185. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 185. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 185.

[0883] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 186. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 186. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 186. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 186. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 186.

[0884] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 187. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 187. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 187. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino

[0885] 88

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[0887] acid sequence set forth in SEQ ID NO: 187. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 187.

[0888] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 188. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 188. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 188. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 188. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 188.

[0889] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 189. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 189. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 189. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 189. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 189.

[0890] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 190. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 190. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 190. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 190. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 190.

[0891] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 191. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 191. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 191. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 191. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 191.

[0892] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 445. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 445. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID 89

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[0894] NO: 445. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 445. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 445.

[0895] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 446. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 446. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 446. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 446. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 446.

[0896] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 447. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 447. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 447. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 447. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 447.

[0897] In some embodiments, a masking peptide comprises at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 449. In some embodiments, a masking peptide comprises at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 449. In some embodiments, a masking peptide comprises at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 449. In some embodiments, a masking peptide comprises at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 449. In some embodiments, a masking peptide comprises at least 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 449.

[0898] In some embodiments, the masking peptide inhibits binding of anti-CD3 to CD3. In some embodiments, provided herein are exemplary masking peptides set forth in Table 8.

[0899] TABLE 8: Exemplary Masking Peptides

[0900] SEQ ID Description Sequence

[0901] NO

[0902] 174 Anti-CD3 idiotype #1 RASENIDSYLA

[0903] CDR-L1

[0904] 175 Anti-CD3 idiotype #1 AATFLAD

[0905] CDR-L2

[0906] 176 Anti-CD3 idiotype #1 QHYYSTPYT

[0907]

[0908] CDR-L3

[0909] 90

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[0911] 177 Anti-CD3 idiotype #1 SYGVS

[0912] CDR-H1

[0913] 178 Anti-CD3 idiotype #1 IIWGDGSTNYHSALIS

[0914] CDR-H2

[0915] 179 Anti-CD3 idiotype #1 GITTVVDDYYAMDY

[0916] CDR-H3

[0917] 180 Anti-CD3 idiotype RASKSVSTSNYSYIH

[0918] (4.15.64) CDR-L1

[0919] 181 Anti-CD3 idiotype YVSYLE

[0920] (4.15.64) CDR-L2

[0921] 182 Anti-CD3 idiotype QHSREFPWT

[0922] (4.15.64) CDR-L3

[0923] 183 Anti-CD3 idiotype DYSIH

[0924] (4.15.64) CDR-H1

[0925] 184 Anti-CD3 idiotype WINTETGEPAYADDFKG

[0926] (4.15.64) CDR-H2

[0927] 185 Anti-CD3 idiotype PYDYDVLDY

[0928] (4.15.64) CDR-H3

[0929] 186 Anti-CD3 idiotype RASENIDSYLA

[0930] (4.32.63) CDR-L1

[0931] 187 Anti-CD3 idiotype AATFLAD

[0932] (4.32.63) CDR-L2

[0933] 188 Anti-CD3 idiotype QHYYSTPYT

[0934] (4.32.63) CDR-L3

[0935] 189 Anti-CD3 idiotype SYGVS

[0936] (4.32.63) CDR-H1

[0937] 190 Anti-CD3 idiotype IIWGDGSTNYHSALIS

[0938] (4.32.63) CDR-H2

[0939] 191 Anti-CD3 idiotype GITTVVDDYYAMDY

[0940] (4.32.63) CDR-H3

[0941] 445 TACT1 Masking Peptide VELTSPLDEN

[0942] 446 TACT2 Masking Peptide GGSCVELTSPLDENC

[0943] 447 TACT3 Masking Peptide QGASTSVDELQAEVDQLEDENYALKTKVAQLRKKVEKL

[0944]

[0945] 449 TACT4 Masking Peptide CTRPDDPEC

[0946] Cleavable Peptide

[0947] The present disclosure provides activatable antibodies comprising one or more cleavable peptides (also referred to as a protease-cleavable peptide or cleavable sequence). A cleavable peptide is a substrate for a protease associated with an autoimmune or inflammatory disease tissue. In some embodiments, the activatable antibody of the disclosure is unable to bind the target until the cleavable peptide is cleaved by a protease in an autoimmune or inflammatory disease tissue of interest. A “cleavage site” as used herein, refers to a recognizable site for protease-mediated cleavage of a peptide. Thus, a cleavage site may be found in the sequence of a cleavable peptide as described herein, including embodiments thereof. In some embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by a protease.

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[0950] To identify enzymes (e.g., proteases) of interest in a particular autoimmune or inflammatory disease tissue, a list of enzymes expressed specifically in the diseased tissue is identified and cleavable peptides are designed based on the enzymes of interest. Candidate cleavable peptides are subsequently screened using a two-pronged approach; 1) sequences are assessed for specificity to one or more enzymes in the desired tissue (positive screen), 2) sequences are assessed for lack of specificity to enzymes in other tissues, such as blood (negative counter screen). The protease-cleavable sequences are further refined to reduce T- and B-cell immunogenicity prediction scores.

[0951] In some embodiments, the cleavable peptide is configured to exhibit reduced cleavage in blood, serum, or plasma while remaining cleavable in autoimmune or inflammatory target tissue environments. Selectivity may be assessed by incubating the activatable antibody in (i) serum or plasma under physiological conditions and (ii) target-tissue-relevant samples (e.g., tissue homogenates, tissue extracts, diseased tissue supernatants, or target-tissue protease mixtures) and measuring cleavage, restoration of binding, and / or restoration of functional activity. Cleavage may be assessed by analytical methods including SDS-PAGE, LC-MS, HPLC / SEC, or capillary electrophoresis, and functional restoration may be assessed by binding assays (e.g., ELISA, BLI / SPR) and cell-based assays.

[0952] In certain embodiments, cleavage of the cleavable peptide restores an immunomodulatory function of the antibody within the target tissue. Non-limiting examples include downmodulation of the TCR complex, induction of T cell anergy or apoptosis, increased Treg abundance or function relative to effector T cells, reduced inflammatory cytokine production, altered trafficking of immune cells, and induction of immune tolerance. Such functional outcomes may be measured using cell-based assays, flow cytometry, cytokine profiling, and tissue-relevant readouts.

[0953] In some embodiments, the cleavable peptide for use in activatable antibodies of the disclosure comprises a consensus sequence. A consensus sequence represents a generalized peptide motif that includes key residues already determined to be essential for recognition and cleavage by a specific protease. These key residues are fixed in the sequence and provide the structural anchors necessary for protease binding and activity. The presence of the letter X at certain positions within the sequence indicates flexibility, allowing various amino acids to occupy those positions without disrupting protease recognition. When designing full cleavage peptides from consensus sequences, each X is replaced with an amino acid selected for one or more objectives:

[0954] 1) Enhancing specific recognition or affinity for the desired protease;

[0955] 2) Minimizing off-target protease activity; and / or

[0956] 3) Facilitating complementary multi-protease targeting.

[0957] In some embodiments, the cleavable peptide is comprised of about 15–20 amino acids. In some embodiments, the cleavable peptide is comprised of less than 50 amino acids. In some embodiments, the 92

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[0959] cleavable peptide is comprised of less than 30 amino acids. In some embodiments, the cleavable peptide is comprised of less than 20 amino acids. In some embodiments, the cleavable peptide is comprised of less than 10 amino acids.

[0960] In some embodiments, the cleavable peptide is a substrate for a protease enriched in an autoimmune or inflammatory diseased tissue. In some embodiments, the cleavable peptide is a substrate for one or more of the following proteases: trypsin, pancreatic lipase, chymotrypsin, elastase, amylase, carboxypeptidase, phospholipase A2, glucokinase, insulin-degrading enzyme (IDE), alpha- 1 -antitrypsin, glutamate dehydrogenase.

[0961] In some embodiments, the cleavable peptide is a substrate for a protease present (e.g., enriched) in the pancreas of a subject with type 1 diabetes. In some embodiments, the cleavable peptide is a substrate for one or more of the following proteases: trypsin and chymotrypsin. In some embodiments, the cleavable peptide is a substrate for trypsin. In some embodiments, the cleavable peptide comprises one or more of the amino acid consensus sequences set forth in Table 9. In some embodiments, the cleavable peptide comprises one or more of the nucleic acid consensus sequences set forth in SEQ ID NOs: 451-469. In some embodiments, the cleavable peptide comprises one or more of the amino acid sequence set forth in Table 10. In some embodiments, the cleavable peptide comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 192-233, 448 or 450.

[0962] In some embodiments, the cleavable peptide is a substrate for a protease present (e.g., enriched) in a joint of a subject with rheumatoid arthritis. In some embodiments, the cleavable peptide is a substrate for one or more of the following proteases: ADAMTS-4 and MMP-13. In some embodiments, the cleavable peptide is a substrate for ADAMTS-5. In some embodiments, the cleavable peptide comprises one or more of the amino acid consensus sequences set forth in Table 11. In some embodiments, the cleavable peptide comprises one or more of the nucleic acid consensus sequences set forth in SEQ ID NOs: 470-483, and 597-601. In some embodiments, the cleavable peptide comprises one or more of the amino acid sequence set forth in Table 12. In some embodiments, the cleavable peptide comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 234-298, and 549-596.

[0963] In some embodiments, the cleavable peptide is a substrate for a protease present (e.g., enriched) in intestinal tissue of a subject with inflammatory bowel disease. In some embodiments, the cleavable peptide is a substrate for one or more of the following proteases: MMP-3, Matripase (ST14), Cathepsin S and Hepsin. In some embodiments, the cleavable peptide is a substrate for urokinase (uPA). In some embodiments, the cleavable peptide comprises one or more of the amino acid consensus sequences set forth in Table 13. In some embodiments, the cleavable peptide comprises one or more of the nucleic acid consensus sequences set forth in SEQ ID NOs: 484-515. In some embodiments, the cleavable peptide

[0964] 93

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[0966] comprises one or more of the amino acid sequences set forth in Table 14. In some embodiments, the cleavable peptide comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 299-381.

[0967] In some embodiments, the cleavable peptide is a substrate for a protease present (e.g., enriched) in the brain of a subject with multiple sclerosis. In some embodiments, the cleavable peptide is a substrate for Capthesin S. In some embodiments, the cleavable peptide comprises one or more of the nucleic acid consensus sequences set forth in Table 15. In some embodiments, the cleavable peptide comprises one or more of the nucleic acid consensus sequences set forth in SEQ ID NOs: 516-530. In some embodiments, the cleavable peptide comprises one or more of the amino acid sequences set forth in Table 16. In some embodiments, the cleavable peptide comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 382-394.

[0968] In some embodiments, the cleavable peptide is a substrate for a protease present (e.g., enriched) in liver of a subject with primary biliary cholangitis. In some embodiments, the cleavable peptide is a substrate for a protease enriched in the liver. In some embodiments, the cleavable peptide is a substrate for hepsin. In some embodiments, the cleavable peptide comprises one or more of the amino acid sequences set forth in Table 17. In some embodiments, the cleavable peptide comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 395-412.

[0969] In some embodiments, the cleavable peptide is a substrate for a protease present (e.g., enriched) in the kidney of a subject with lupus nephritis. In some embodiments, the cleavable peptide is a substrate for a protease present (e.g., enriched) in kidney of a subject receiving an allogeneic kidney transplant. In some embodiments, the cleavable peptide is a substrate for a protease enriched in a diseased kidney. In some embodiments, the cleavable peptide is a substrate for one or more of the following proteases:

[0970] Meprin, Meprin B, or Renin. In some embodiments, the cleavable peptide comprises one or more of the amino acid sequences set forth in Table 18. In some embodiments, the cleavable peptide comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 413-435.

[0971] In some embodiments, the cleavable peptide is a substrate for a protease present (e.g., enriched) in the eye of a subject with Sjorgen’s syndrome-associated dry eye or autoimmune optic neuritis.

[0972] TABLE 9: Consensus Pancreas-Specific Cleavable Peptide Sequences

[0973] Consensus Sequence Amino Acid Substitutions

[0974] K-xl-x2-x3-R-x4-x5-x6-Y-x7-x8-x9-W xl-x3=A, S, T, G, x4-x6 = E, D, G, x7-x9 = V, I, A, L K-xl-x2-x3-R-x4-x5-x6-Y-x7-x8-x9-F xl-x3 = A, S, T, G, x4-x6 = D, E, G, x7-x9 = V, I, A, F K-x 1 -x2-x3-R-x4-x5- W-x6-x7 - Y xl-x3 = G, T, S, x4-x5 = G, E, D, x6-x7 = A, L

[0975] K-xl-x2-x3-R-x4-x5-x6-F-x7-x8-x9-Y xl-x3 = A, G, T, x4-x6 = E, D, x7-x9 = I, V, A

[0976] R-xl-x2-x3-K-x4-x5-x6-F-x7-x8-x9-Y xl-x3 = A, S, T, x4-x6 = D, E, G, x7-x9 = V, I, L

[0977]

[0978] K-x 1 -x2-x3-R-x4-x5- Y-x6-x7 -F xl-x3 = A, G, S, x4-x5 = E, D, x6-x7 = V, I

[0979] 94

[0980] FH13277368.1Attorney Docket No: TGY-00125

[0981] K-xl-x2-x3-R-x4-x5-F-x6-x7-Y xl-x3 = G, A, T, x4-x5 = G, D, x6-x7 = I, V

[0982] R-xl-x2-x3-K-x4-x5-x6-F-x7-x8-x9-L xl-x3 = A, T, S, x4-x6 = D, E, x7-x9 = I, L, V

[0983] K-xl-x2-R-x3-x4-F-x5-x6-L xl-x2 = G, A, T, x3-x4 = E, D, x5-x6 = L, I

[0984] K-xl-x2-R-x3-x4-F-x5-x6-Y xl-x2 = G, A, x3-x4 = D, E, x5-x6 = V, I

[0985] K-xl-x2-x3-R-x4-x5-x6-L-x7-x8-x9-F xl-x3 = A, G, T, x4-x6 = D, E, x7-x9 = V, I

[0986] K-xl-x2-x3-R-x4-x5-x6-F xl-x3 = G, A, x4-x5 = E, D

[0987] R-xl-x2-x3-K-x4-x5-x6-Y xl-x3 =A, S, T, x4-x6 = D, G

[0988] K-xl-x2-x3-R-x4-x5-x6-Y xl-x3 = G, A, T, x4-x5 = E, D

[0989] K-xl-x2-x3-R-x4-x5-x6-W xl-x3 = A, G, S, x4-x5 = E, D

[0990] R-xl-x2-x3-K-x4-x5-x6-F xl-x3 = G, A, T, x4-x5 = E, D

[0991] K-xl-x2-x3-R-x4-x5-x6-L-x7-x8-x9-Y xl-x3 = A, S, T, x4-x6 = G, D, x7-x9 = V, I

[0992] R-xl-x2-x3-K-x4-x5-x6-F-x7-x8-x9-V xl-x3 = G, A, T, x4-x5 = D, E, x7-x9 = I, V

[0993]

[0994] R-xl-x2-x3-K-x4-x5-x6-I-x7-x8-x9-F xl-x3 = G, A, T, x4-x5 = E, D, x7-x9 = L, I

[0995] TABLE 10: Exemplary Pancreas-Specific Cleavable Peptides

[0996] KLSRKGFYVW KLSRKGFYVF KLARGFYF KTSRRGFYRW KLSRKGFYFV KTARKGFAL KLSRKGFLNF KLSRRGFYVW KLARAGFYV KLSRKGFYWF KTSRRGFYRW KSSRKGYYWF KTSRKGFYFY KLSRKGFYVF KSSRLGFLMF KLSRKGFYRY KLSRKGFLNF RNSRLGIMLF KTSRKGFYLY KTARKGFAL RASRKGFAVL KLSRKGFYWL KLARAGFYV KQTRAGFYS K KGFYWF KLSRKGFLY KQSRLGFTS KLSRKGFWYF KLSRKGFLY KPFRRGFYS KLSRRGFYLF KLARGFYV KQTRLGFYS KTSRKGFYVW KTARKGFLS KQTRAGFYS KSSRRGFLY KASRKGFLY RGSKKFTAL

[0997]

[0998] KTSRKGFLY KTSRGFAL KQTRAGFAS

[0999] TABLE 11: Consensus Joint-Specific Cleavable Peptide Sequences

[1000] Consensus Sequence Amino Acid Substitutions

[1001] P-xl-F-x2-G-x3-H-x4-Q xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3= A, G, L, P, Q, x4= A, G,

[1002] L, P, Q

[1003] P-xl-F-x2-G-x3-L-x4-A xl = G, T, A, x2 = G, S, x3 = S, G, x4 = L, A

[1004] P-xl-F-x2-G-x3-Q-x4-A xl= A, G, L, P, Q, x2=A, G, L, F, Q, x3= A, G, L, P, Q, x4= A, G,

[1005] L, P, Q

[1006] P-xl-F-x2-G-x3-S-x4-L xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3= A, G, L, P, Q, x4= A, G,

[1007] L, P, Q

[1008] P-xl-F-x2-G-x3-V-x4-L-x5-A xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3= A, G, L, P, Q, x4= A, G,

[1009] L, P, Q, x5= A, G, L, P, Q

[1010] P-x 1 -L-x2-G-x3-Q-x4-N xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3=A, G, L, P, Q, x4= A, G,

[1011]

[1012] L, PQ

[1013] 95

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[1015] P-xl-T-x2-G-x3-Q-x4-L xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3= A, G, L, P, Q, x4= A, G,

[1016] L, P, Q

[1017] Q-xl-F-x2-G-x3-N-x4-L xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3= A, G, L, P, Q, x4= A, G,

[1018] L, P, Q

[1019] Q-xl-F-x2-G-x3-T-x4-N xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3= A, G, L, P, Q, x4= A, G,

[1020] L, P, Q

[1021] Q-xl-L-x2-G-x3-S-x4-L xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3= A, G, L, P, Q, x4= A, G,

[1022] L, P, Q

[1023] R-xl-x2-H-x3-x4-N xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3= A, G, L, P, Q, x4= A, G,

[1024] L, P, Q

[1025] R-xl-F-x2-G-x3-x4-N xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3= A, G, L, P, Q, x4= A, G,

[1026] L, P, Q

[1027] R-xl-F-x2-G-x3-V-x4-L xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3= A, G, L, P, Q, x4= A, G,

[1028] L, P, Q

[1029] R-x 1 -F-x2-L-x3- A xl= A, G, L, P, Q, x2= A, G, L, P, Q, x3= A, G, L, P, Q, x4= A, G,

[1030] L, P, Q

[1031] xl-x2-N-I-T-E-G-E-A-R-G-S- xl-x2 = D, E, N, Q, S, T, x3 = A, N, Q, S, T

[1032] V-I-x3

[1033] xl-x2-N-I-T-E-G-E-A-R-G-S- xl-x2 = D, E, N, Q, S, T, x3 = D, E, N, Q, S, T

[1034] x3

[1035] xl-x2-T-E-G-E-A-R-G-S-V-I- xl-x2 = D, E, N, Q, S, T, x3 = A, D, E, N, Q, S, T

[1036] x3

[1037] x 1 -x2-x3-N-I-T-E-G-E- A-R- xl-x3 = P, Q, G; x4 = G, N, S; x5-x6 = P, Q, G, N, S

[1038] x4-x5-x6

[1039] x 1 -P-x2-N-I-T-E-G-E- A-R-x3- xl = A, G, N, Q, S, x2 = A, G, N, Q, S, x3 = A, G, N, Q, S, x4 =

[1040]

[1041] P-x4 A, G, N, Q, S

[1042] TABLE 12: Exemplary Joint-Specific Cleavable Peptides

[1043] PAFAGAHAQ PPLPGPQPN QQLQGQSQL ENITEGEARGSVI PAFAGAQAA PPTPGPQPL RAAHAAN QNITEGEARGSVI PAFAGASAL PQFQGQHQQ RAFAGAAN DNITEGEARGSVIN PAFAGAVALAA PQFQGQQQA RAFAGAVAL SNITEGEARGSVIN PALAGAQAN PQFQGQSQL RAFALAA ENITEGEARGSVIQ PATAGAQAL PQFQGQVQLQA RGFGGGGN QNITEGEARGSVIN PGFGGGHGQ PQLQGQQQN RGFGGGVGL QNITEGEARGSVIT PGFGGGQGA PQTQGQQQL RGFGLGA ENITEGEARGSVIT PGFGGGSGL QAFAGANAL RGGHGGN DNITEGEARGSVIS PGFGGGVGLGA QAFAGATAN RLFLGLLN DNTEGEARGSVINQ PGLGGGQGN QALAGASAL RLFLGLVLL DNTEGEARGSVIE PGTGGGQGL QGFGGGNGL RLFLLLA QNTEGEARGSVIN PLFLGLHLQ QGFGGGTGN RLLHLLN QNTEGEARGSVINS PLFLGLQLA QGLGGGSGL RPFPGPPN SNTEGEARGSVIT PLFLGLSLL QLFLGLNLL RPFPGPVPL SNTEGEARGSVINT PLFLGLVLLLA QLFLGLTLN RPFPLPA PQQNITEGEARGNP QLLLGLSLL

[1044]

[1045] PLLLGLQLN RPPHPPN PPGNITEGEARGQQ 96

[1046] FH13277368.1Attorney Docket No: TGY-00125

[1047] PLTLGLQLL QPFPGPNPL RQFQGQQN GQPNITEGEARNGS PPFPGPHPQ QPFPGPTPN RQFQGQVQL QGPNITEGEARSGQ PPFPGPQPA QPLPGPSPL RQFQLQA QPGNITEGEARNPQ PPFPGPSPL QQFQGQNQL RQQHQQN NPGNITEGEARGPQ PPFPGPVPLPA QQFQGQTQN DNITEGEARGSVI QPGNITEGEARNPQ QPGNITEGEARGNS APGNITEGEARGNV PGQNITEGEARSGV GQQNITEGEARNGI PGNITEGEARGNVI QGNITEGEARGNVL QQNITEGEARGNVL GPNITEGEARGNLI APQNITEGEARSPG GPNITEGEARNSP DNITEGEARGSE ENITEGEARGSD QNITEGEARGSE NITEGEARGNSVI NITEGEARGNSVL IITEGEARNNLIL DNITEGEARGSVIN ENITEGEARGSVIN QNITEGEARGSVIT QPGNITEGEARGSVI PQQNITEGEARGSVIN GQPNITEGEARGSVIT DNITEGEARGSEE QNITEGEARGSDE

[1048]

[1049] TABLE 13: Consensus Intestine-Specific Cleavable Peptide Sequences

[1050] Consensus Sequence Amino Acid Substitutions

[1051] P-xl-A-x2-G-x3-L xl = A, G, V, x2 = T, S, A, x3 = G, S

[1052] P-xl-Q-x2-G-x3-L xl = A, S, V, x2 = N, T, S, x3 = G, A

[1053] P-xl-L-x2-G-x3-F xl = V, A, L, x2 = G, T, S, x3 = S, A

[1054] P-xl-V-x2-G-x3-L xl = A, V, L, x2 = G, T, A, x3 = S, G

[1055] P-xl-A-x2-G-x3-L-x4-Y-x5-V xl = G, A, V, x2 = T, S, A, x3 = S, G, x4 = F, Y, x5 = L, I

[1056] P-xl-Q-x2-G-x3-L-x4-F-x5-S xl = S, A, G, x2 = N, T, A, x3 = G, S, x4 = Y, F, x5 = T, S

[1057] P-xl-L-x2-G-x3-F-x4-A-x5-T xl = A, V, L, x2 = G, S, T, x3 = S, A, x4 = T, S, A, x5 = G, T P-x 1 - V-x2-G-x3-L-x4-W-x5 - A xl = G, V, L, x2 = A, S, G, x3 = S, G, x4 = Y, W, x5 = A, G xl-L-x2 xl= G, V, S, A, x2= F, P, G, T, A, Y, G

[1058] G-xl-F xl= L

[1059] A-xl-L-x2 xl= G, x2= S

[1060] xl-L-F xl=G

[1061] A-L-xl-S xl=G

[1062] xl-L-x2-V xl— G, x2= A

[1063] S-L-F-xl xl=A

[1064] A-xl-L-F xl=G

[1065] P-L-S-xl xl=G

[1066] S-xl-L-F xl=V

[1067] xl-L-V-x2 xl= G, T, x2= S, G

[1068] A-L-xl-F xl=S

[1069] T-L-V-xl xl=G

[1070] S-L-xl-F xl=G

[1071]

[1072] xl-V-L-x2 xl— G, x2= A

[1073] TABLE 14: Exemplary Intestine-Specific Cleavable Peptides

[1074] PSAGILYGVDV PVQGILFSTGS PVGVALWVAGA PSTGILYGVEV PQQGILFSTAS PAVGSLWVTGA

[1075]

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[1078] PSAGILYGVDI PGLGFSLTGTT PVAGSLWVGTA PSAGVLFGVDV PGIGFSLTGLT PVVGSLWVATA PSAGILFGLDV PGLGFSLTATV PSAGVLI PSTGILYGVDV PLGGFSLTGST PSAGVIL PSGGILYGVTV PGLGFSITGTT PSAGVLW PAQGILFSTAS PGIGFSLTGAT PSIGVLI PVQGILFSTIS PGIGFSLTTVT PSAGVVI PAQGILFATAS PAVGSLWVAGA PSGGVIL PAQGILFSTGV PVAGSLWVAGA PSAVVI PAQGVLFSTAS PAVGSLWVAGG RLSGFGL RPSGGGL RQPGKGL RVSGKGL RQSGTGL RLSGVGL RSPGYGL RKSGFGL RSPGKGL RKSGYGL RLSGRGL RAPGVGL RPSGTGL RQPGAGL RKPGTGL RQSGKGL RPSGFGL RQPGYGL GAP AGLS ALSF GLA GLF TLVG GLP ALGS SLGF GLT GLAV GVLA GLY SLFA VLA GVP AGLF VLP SLA PLSG VLG SLG SVLF VLT ALG GLVS VGA RRKVWF GRRVRW GRKVKWF AKRKVDL GDRRSRW DRRKRVW GARRRV KKRSRWL GKRKVDL SKRGRWL DRRGRV ARGRVW DRKVKFL GKRVRWL DRRRWL GRRGRWF GKRGRVF DKRSRLF

[1079]

[1080] TABLE 15: Consensus Brain-Specific Cleavable Peptide Sequences

[1081] Consensus Sequence Amino Acid Substitutions

[1082] xl-L-x2 xl= G, V, S, A, x2= F, P, G, T, A, Y, G

[1083] G-xl-F xl= L

[1084] A-xl-L-x2 xl= G, x2= S

[1085] xl-L-F xl=G

[1086] A-L-xl-S xl=G

[1087] xl-L-x2-V xl— G, x2= A

[1088]

[1089] S-L-F-xl xl=A

[1090] 98

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[1092] A-xl-L-F xl=G

[1093] P-L-S-xl xl=G

[1094] S-xl-L-F xl=V

[1095] xl-L-V-x2 xl= G, T, x2= S, G

[1096] A-L-xl-F xl=S

[1097] T-L-V-xl xl=G

[1098] S-L-xl-F xl=G

[1099]

[1100] xl-V-L-x2 xl— G, x2= A

[1101] TABLE 16: Exemplary Brain-Specific Cleavable Peptides

[1102] AGLS ALSF GAP GLF TLVG GLA ALGS SLGF GLP GLAV GVLA GLT SLFA VLA GLY AGLF VLP GVP PLSG VLG SLA SVLF VLT SLG

[1103]

[1104] GLVS VGA ALG

[1105] TABLE 17: Exemplary Liver-Specific Cleavable Peptides

[1106] GRRVRW GRKVKWF RRKVWF GDRRSRW DRRKRVW AKRKVDL KKRSRWL GKRKVDL GARRRV DRRGRV ARGRVW SKRGRWL GKRVRWL DRRRWL DRKVKFL GKRGRVF DKRSRLF GRRGRWF

[1107]

[1108] TABLE 18: Exemplary Kidney-Specific Cleavable Peptides

[1109] DLMEQQV EFIDAL DFMEQNL NLTDEQ ETLDAL DLFEQNR ELYEEDT EFTDAL DLMEQNV DLMEQEV NLGDEQ ELFEEDT DLFEQEY NFGDEQ ELIEEDR DFMEQEV NLFDEQ ETFEEDL DLMEQEG NLIDEQ EFVEEDN ELTDAL DLMEQNQ

[1110]

[1111] 99

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[1113] Linker Peptide

[1114] The present disclosure provides linker peptides for use in the activatable antibodies of the disclosure. In some embodiments, a linker peptide provides flexibility and spacing between a masking peptide and a binding domain, e.g., a spacer linker. In some embodiments, a linker peptide comprises a flexible linker including one or more glycine residues, serine residues, alanine residues, histidine residues, and / or proline residues. In some embodiments, a linker peptide links the masking peptide to a cleavable peptide or a binding domain, or both, in an activatable antibody.

[1115] In some embodiments, a linker peptide comprises 1-30 amino acids. In some embodiments, a linker peptide comprises less than 30 amino acids. In some embodiments, a linker peptide comprises less than 20 amino acids. In some embodiments, a linker peptide comprises less than 10 amino acids.

[1116] In some embodiments, a masking peptide is linked to an N-terminus of a light chain or heavy chain of the activatable antibody via a linker comprising a cleavable peptide. In some embodiments, the masking peptide is linked to an N-terminus of a light chain variable region or heavy chain variable region of an activatable antibody via a linker comprising a cleavable peptide. In some embodiments, a masking peptide is linked to a C-terminus of a light chain or heavy chain of the activatable antibody via a linker comprising a cleavable peptide. In some embodiments, a masking peptide is linked to a C-terminus of a light chain variable region or heavy chain variable region of the activatable antibody via a linker comprising a cleavable peptide.

[1117] In some embodiments, an activatable antibody of the disclosure comprises more than one linkers, e.g., a first spacer linker and a second spacer linker. In some embodiments, an activatable antibody comprises a linker comprising a cleavable peptide.

[1118] In some embodiments, a linker comprising a cleavable peptide comprises, in an amino-terminus to carboxy-terminus direction: a spacer linker, a cleavable peptide, and a spacer linker. In some embodiments, a C-terminus of the masking peptide is linked to an N-terminus of the linker comprising a cleavable peptide, and a C-terminus of the linker comprising a cleavable peptide is linked to an N-terminus of the first chain, e.g., a light chain or a light chain variable region.

[1119] In some embodiments, an activatable antibody comprises a linker selected from Ala linker, Gly linker, and GlySer linker. In some embodiments, the linker is a GlySer linker set forth in SEQ ID NO: 436 (GGGGS). In some embodiments, the linker is a GlySer linker set forth in SEQ ID NO: 437 ((G4S)5). In some embodiments, the linker is a Gly Ala linker set forth in SEQ ID NO: 438 (GGGGA).

[1120] In some embodiments, the present disclosure provides a nucleic acid encoding an activatable antibody of the disclosure, wherein the nucleic acid comprises a nucleotide sequence encoding an N-terminus GlySer linker. In some embodiments, a nucleic acid encoding an activatable antibody comprises a nucleic acid

[1121] 100

[1122] FH13277368.1Attorney Docket No: TGY-00125

[1123] encoding a C -terminus GlySer linker. In some embodiments, a nucleic acid encoding an activatable antibody comprises a nucleic acid sequence encoding an N-terminus and C-terminus GlySer linker.

[1124] Bifunctional Activatable Antibodies

[1125] In some embodiments, the activatable antibody further comprises an additional functional domain distinct from the first antibody binding domain (e.g., anti-CD3, anti-PD-1, anti-TNFR2, anti-CD2). In some embodiments, the second functional domain is an inflammation-regulating peptide. In some embodiments, the second functional domain is a second antigen binding domain.

[1126] In some embodiments, the activatable antibody comprises an inflammation-regulating peptide. As used herein, an inflammation-regulating peptide refers to a peptide or peptide-derived moiety that, upon local release or activation within diseased tissue, modulates inflammatory or immune responses to promote immune homeostasis, including attenuation of inflammatory signaling and / or induction of regulatory or tolerogenic pathways.

[1127] In some embodiments, the masking domain further comprises an inflammation-regulating peptide (IRP), such that conditional activation results in local release or exposure of the inflammation-regulating peptide and attenuation of inflammatory signaling and / or promotion of regulatory or tolerogenic pathways within the inflamed tissue microenvironment. In some embodiments, the activatable antibody mediates target engagement following localized activation after protease-mediated cleavage, and the inflammation-regulating peptide targets a distinct biological pathway. In some embodiments, the inflammationregulating peptide is integrated as part of the masking domain and / or linked via a linker peptide to the Fc domain, or directly fused to the Fc domain (FIG. 18). In some embodiments, the inflammation-regulating peptide is linked to the masking peptide. In some embodiments, the inflammation-regulating peptide is linked to the Fc domain of the activatable antibody. In some embodiments, the inflammation-regulating peptide is linked to the VH domain of the activatable antibody. In some embodiments, the inflammationregulating peptide is linked to the VL domain of the activatable antibody. In some embodiments, the inflammation-regulating peptide is linked via a linker peptide. In some embodiments, the linker peptide is a protease-cleavable linker. In some embodiments, the inflammation-regulating peptide is masked.

[1128] Upon conditional activation within diseased tissue, the inflammation-regulating peptide provides a complementary or synergistic local immunomodulatory effect, resulting in localized regulation of inflammatory signaling without inducing systemic immunosuppression or systemic immunoregulation. In some embodiments, the inflammation regulating peptide comprises local cytokine blockade, receptor agonism, or promotion of protective regulatory immune pathways.

[1129] In some embodiments, the inflammation-regulating peptide is linked to the masking domain of an activatable antibody. In some embodiments, the inflammation-regulating peptide comprises the masking 101

[1130] FH13277368.1Attorney Docket No: TGY-00125

[1131] domain. In some embodiments, the inflammation-regulating peptide comprises the masking domain or a fragment thereof. In some embodiments, cleavage or release of the masking domain results in local attenuation of inflammatory cytokine signaling and promotion of immune homeostasis. In some embodiments, the inflammation-regulating peptide exerts a local immunomodulatory effect. Such local immunomodulatory activity may reduce inflammatory cytokine signaling, promote regulatory immune responses, or attenuate excessive immune activation without inducing systemic immunosuppression.

[1132] In some embodiments, the inflammation-regulating peptide comprises a peptide or peptidederived moiety capable of locally modulating immune signaling pathways, comprising dominant-negative cytokine ligands or fragments thereof, cytokine receptor agonists or antagonists, decoy receptor mimetics, or peptides that promote regulatory immune cell activity or immune tolerance. In some embodiments, the inflammation-regulating peptide targets CD3, PD-1, TNFR2, CD25, TNF, IL-6, IL-17, IL- 23, TL1A, TSLP, IL-33, and / or IL-2 variants.

[1133] In some embodiments, the activatable antibody comprises a second antigen binding domain. In some embodiments, an activatable antibody comprises a first antigen binding domain and a second antigen binding domain. In some embodiments, an activatable antibody comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain is anti-CD3 and the second antigen binding domain is anti-TNFR2. In some embodiments, an activatable antibody comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain is anti-PD-1 and the second antigen binding domain is anti-TNFR2. In some embodiments, an activatable antibody comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain is anti-CD3 and the second antigen binding domain is anti-TNFRl. In some embodiments, an activatable antibody comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain is anti-TNFRl and the second antigen binding domain is anti-TNFR2. In some embodiments, an activatable antibody comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain is anti-PD-1 and the second antigen binding domain is anti-TNFR1. In some embodiments, an activatable antibody comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain is anti-CD25 and the second antigen binding domain is anti-TNFRl. In some embodiments, an activatable antibody comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain is anti-CD25 and the sec...

Claims

Attorney Docket No: TGY-00125WHAT IS CLAIMED IS1. An activatable antibody comprising:a) an antibody, or antigen binding fragment thereof, that specifically binds an immunomodulatory T cell molecule;b) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) to the immunomodulatory T cell molecule;c) a cleavable peptide coupled to the masking peptide of (b) and the antibody or antigen binding fragment of (a), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,d) one or more linker peptides linking (a) and (c) and / or (b) and (c).

2. The activatable antibody of claim 1, wherein the immunomodulatory T cell molecule modulates one or more of the following properties:a) downregulation and / or inhibition of a CD3 / T Cell Receptor (TCR) complex;b) activation of effector T cells (Teffs);c) activation of regulatory T cells (Tregs);d) T cell trafficking;e) immune tolerance; andf) a combination of (a)-(e).

3. The activatable antibody of claim 2, wherein the activatable antibody inhibits the immunomodulatory T cell molecule and / or activates Tregsand / or enhances immune suppression, and / or inhibits effector T cells.

4. The activatable antibody of claim 2, wherein the activatable antibody stimulates the immunomodulatory T cell molecule and / or activates Tregsand / or enhances immune suppression, and / or inhibits effector T cells.

5. The activatable antibody of claim 2, wherein the activatable antibody inhibits interaction of the immunomodulatory T cell molecule with one or more endogenous ligands to and / or inhibits effector T cells.

6. The activatable antibody of claim 1, wherein the immunomodulatory T cell molecule is selected from a cytokine receptor, a chemokine receptor, an immune checkpoint, a member of the Tumor Necrosis 235FH13277368.1Attorney Docket No: TGY-00125Factor (TNF) superfamily, a member of the Immunoglobulin Superfamily (IgSF), an integrin, or an adhesion molecule.

7. The activatable antibody of claim 6, wherein the immunomodulatory T cell molecule is selected from CD3, CD28, ICOS, IL-2Ra, IL-2R, IL-7R, IL-10R, PD-1, PD-L1, CTLA-4, TNFR2, CCR4, CCR8, CXCR3, 0X40, OX40L, GITR, TSLPR, CD137 (4-1BB), CD40L (CD154), LAG-3 (CD223), TIM-3 (HAVCR2), VISTA, TIGIT, GARP, LAP, NRP1, BTLA, CD27, CD39, CD47, CD73, CD276, 2B4 (CD244), αEβ7, CD62L, LFA-1, ICAM-1, or a combination thereof.

8. The activatable antibody of any of claims 1-7, wherein the immunomodulatory T cell molecule is expressed on Tregs, effector T cells, or both Tregsand effector T cells.

9. The activatable antibody of any one of claims 1-8, wherein the autoimmune or inflammatory disease is selected from type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, primary biliary cholangitis, lupus nephritis, Sjorgen’s syndrome- associated dry eye, and autoimmune optic neuritis.

10. The activatable antibody of any one of claims 1-9, wherein the autoimmune or inflammatory disease tissue is selected from pancreas, joint, intestine, brain, liver, kidneys, heart and eyes.

11. The activatable antibody of any one of claims 1-10, wherein the masking peptide comprises an amino acid sequence which binds to one or more CDRs of the antibody or antigen binding fragment of (a).

12. The activatable antibody of any one of claims 1-10, wherein the masking peptide is an antiidiotype antibody or an antigen binding fragment thereof.

13. The activatable antibody of any one of claims 1-10, wherein the masking peptide is identified by phage display, in vivo immunization, or in silico using artificial-intelligence (Al)-based tools.

14. The activatable antibody of any one of the preceding claims, wherein the cleavable peptide is a substrate for a protease in the disease tissue.236FH13277368.1Attorney Docket No: TGY-0012515. The activatable antibody of any one of the preceding claims, wherein the cleavable peptide comprises a consensus amino acid sequence recognized by the protease in the disease tissue.

16. The activatable antibody of any one of the preceding claims, wherein the cleavable peptide is less than 50 amino acids, less than 30 amino acids, less than 20 amino acids or less than 10 amino acids.

17. The activatable antibody of any one of claims 1-16, wherein the cleavable peptide comprises a consensus amino acid sequence set forth in Table 9, Table 11, Table 13, or Table 15, or a variant thereof.

18. The activatable antibody of any one of claims 1-16, wherein the cleavable peptide comprises an amino acid sequence set forth in Table 10, Table 12, Table 14, Table 16, Table 17 or Table 18, or a variant thereof.

19. An activatable antibody that specifically binds CD3 epsilon (CD3ε) comprising:a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 1-3 or SEQ ID NOs: 7-9;b) a heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 4-6 or SEQ ID NOs: 10-12;c) a masking peptide which binds to and inhibits the binding of the antibody to CD3ε;d) a cleavable peptide coupled to the masking peptide and either the VL or VH region of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue: and, optionally,e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

20. The activatable antibody of claim 19, comprising:a) a VL region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs:1-3; and a VH region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 4-6; orb) a VL region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs:7-9; and a VH region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 10-12.237FH13277368.1Attorney Docket No: TGY-0012521. The activatable antibody of claim 19, comprising a light chain variable (VL) region comprising the sequence of SEQ ID NO: 13 and heavy chain variable (VH) region comprising the sequence of SEQ ID NO: 14.

22. The activatable antibody in any one of claims 19 -21, comprising a human IgG Fc region which binds Fc receptors.

23. The activatable antibody of any one of claims 19-22, wherein the masking peptide is selected from an amino acid sequence of SEQ ID NOs: 174-191.

24. The activatable antibody of any one of claims 19-23, wherein binding to CD3s induces downregulation and / or inhibition of the TCR complex, and / or effector T cell death, and / or expansion and / or proliferation of Tregs, and / or cytokine production, or a combination thereof.

25. An activatable antibody that specifically binds PD- 1, comprising:a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 15-17;b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 18-20;c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD-1;d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

26. An activatable antibody that specifically binds PD- 1, comprising:a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 21-23;b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 24-26;c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD-1;238FH13277368.1Attorney Docket No: TGY-00125d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

27. An activatable antibody that specifically binds PD- 1, comprising:a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 27-29;b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 30-32;c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD-1;d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

28. An activatable antibody that specifically binds PD- 1, comprising:a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 33-35;b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 36-38;c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD-1;d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

29. An activatable antibody that specifically binds PD-1, comprising:a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 39-41;b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 42-44;239FH13277368.1Attorney Docket No: TGY-00125c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD- 1;d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

30. An activatable antibody that specifically binds PD- 1, comprising:a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 45-47;b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 48-50;c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD-1;d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue: and, optionally,e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

31. An activatable antibody that specifically binds PD- 1, comprising:a) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 51-53;b) heavy chain variable (VH) region comprising the CDR1, CDR2, and CDR3 amino acid sequences of SEQ ID NOs: 54-56;c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to PD- 1;d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

32. The activatable antibody of any one of claims 25-31, wherein binding to PD-1 induces effector T cell death.240FH13277368.1Attorney Docket No: TGY-0012533. An activatable antibody that specifically binds TNFR2 comprising:a) a light chain variable (VL) region comprising a CDR-L1, CDR-L2, and CDR-L3 and heavy chain variable region (VH) comprising a CDR-H1, CDR-H2, and CDR-H3 amino acid sequence set forth in Table 4A;b) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) to TNFR2;c) a cleavable peptide coupled to the masking peptide of (b) and the antibody or antigen binding fragment of (a), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,d) one or more linker peptides linking (a) or (b) with (c), or (b) with (c).

34. An activatable antibody that specifically binds TNFR2 comprising:a) a light chain variable (VL) region comprising a CDR1, CDR2, and CDR3 amino acid sequence set forth in any one of SEQ ID NOs: 106-134;b) a heavy chain variable (VH) region comprising a CDR1, CDR2, and CDR3 amino acid sequence set forth in any one of SEQ ID NOs: 135-171;c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to TNFR2;d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

35. An activatable antibody that specifically binds TNFR2 comprising:a) a polypeptide sequence set forth in any one of SEQ ID NOs: 104-105;b) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to TNFR 2;c) a cleavable peptide coupled to the masking peptide of (b) and the antibody or antigen binding fragment of (a), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,d) one or more linker peptides linking (a) with (c), or (b) with (c).

36. An activatable antibody that specifically binds CD2, comprising:a) heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 172,241FH13277368.1Attorney Docket No: TGY-00125b) a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 173, c) a masking peptide which binds to and inhibits the binding of the antibody or antigen binding fragment of (a) or (b) to CD2;d) a cleavable peptide coupled to the masking peptide of (c) and the antibody or antigen binding fragment of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

37. The activatable antibody of any one of claims 19-36, wherein the activatable antibody modulates one or more of the following properties:a) downregulation and / or inhibition of a CD3 / T Cell Receptor (TCR) complex;b) activation of effector T cells (Teffs);c) activation of regulatory T cells (Tregs);d) T cell trafficking;e) immune tolerance; andf) a combination of (a)-(e).

38. The activatable antibody of any one of claims 19-36, wherein the activatable antibody inhibits CD3, PD-1, TNFR2 and / or CD2 and / or activates Tregsand / or enhances immune suppression, and / or inhibits effector T cells.

39. The activatable antibody of any one of claims 19-36, wherein the activatable antibody stimulates CD3, PD-1, TNFR2 and / or CD2 and / or activates Tregsand / or enhances immune suppression, and / or inhibits effector T cells.

40. The activatable antibody of any one of claims 19-36, wherein the activatable antibody inhibits interaction of CD3, PD-1, TNFR2 and / or CD2 with one or more endogenous ligands to and / or inhibits effector T cells.

41. The activatable antibody of any one of claims 19-36, wherein CD3, PD-1, TNFR2 and / or CD2 is expressed on Tregs, effector T cells, or both Tregsand effector T cells.

42. The activatable antibody of any one of claims 19-41, wherein the autoimmune or inflammatory disease is selected from type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, multiple 242FH13277368.1Attorney Docket No: TGY-00125sclerosis, primary biliary cholangitis, lupus nephritis, Sjorgen’s syndrome- associated dry eye, and autoimmune optic neuritis.

43. The activatable antibody of any one of claims 19-41, wherein the autoimmune or inflammatory disease tissue is selected from pancreas, joint, intestine, brain, liver, kidneys, heart and eyes.

44. The activatable antibody of any one of claims 19-43, wherein the masking peptide comprises an amino acid sequence which binds to one or more CDRs of the antibody or antigen binding fragment of (a).

45. The activatable antibody of any one of claims 19-43, wherein the masking peptide is an antiidiotype antibody or an antigen binding fragment thereof.

46. The activatable antibody of any one of claims 19-45, wherein the masking peptide is identified by phage display, in vivo immunization, or in silico using artificial-intelligence (Al)-based tools.

47. The activatable antibody of any one of claims 19-46, wherein the cleavable peptide is a substrate for a protease in the disease tissue.

48. The activatable antibody of any one of claims 19-47, wherein the cleavable peptide comprises a consensus amino acid sequence recognized by the protease in the disease tissue.

49. The activatable antibody of any one of claims 19-48, wherein the cleavable peptide is less than 50 amino acids, less than 30 amino acids, less than 20 amino acids or less than 10 amino acids.

50. The activatable antibody of any one of claims 19-49, wherein the cleavable peptide comprises a consensus amino acid sequence set forth in Table 9, Table 11, Table 13, or Table 15, or a variant thereof.

51. The activatable antibody of any one of claims 19-49, wherein the cleavable peptide comprises an amino acid sequence set forth in Table 10, Table 12, Table 14, Table 16, Table 17 or Table 18, or a variant thereof.

52. The activatable antibody of claim 1, wherein the immunomodulatory T cell molecule is selected from CD3, CD28, ICOS, IL-2, IL-2Ra, IL-2R, IL-7R, IL-10R, PD-1, PD-L1, CTLA-4, TNFR2, CCR4,243FH13277368.1Attorney Docket No: TGY-00125CCR8, CXCR3, 0X40, OX40L, GITR, TSLPR, CD137 (4-1BB), CD40L (CD154), LAG-3 (CD223), TIM-3 (HAVCR2), VISTA, TIGIT, GARP, LAP, NRP1, BTLA, CD27, CD39, CD47, CD73, CD276, 2B4 (CD244), αEβ7, CD62L, LFA-1, ICAM-1, or a combination thereof.

53. An activatable antibody that specifically binds human CD3 comprising:a) a light chain variable (VL) region;b) a heavy chain variable (VH) region;c) at least one masking peptide which binds to and inhibits the binding of the antibody to CD3a; d) a cleavable peptide coupled to the at least one masking peptide and either the VL or VH region of (a) or (b), wherein the cleavable peptide comprises a substrate for a protease in an autoimmune or inflammatory disease tissue; and, optionally,e) one or more linker peptides linking (a) or (b) with (d), or (c) with (d).

54. The activatable antibody of claim 53, wherein the VL comprises a CDR-L1 set forth in SEQ ID NO: 1, a CDR-L2 set forth in SEQ ID NO: 2, a CDR-L3 set forth in SEQ ID NO: 3, and wherein the VH comprises a CDR-H1 set forth in SEQ ID NO: 4, a CDR-H2 set forth in SEQ ID NO: 5, and a CDR-H3 set forth in SEQ ID NO: 6.

55. The activatable antibody of claim 53, wherein the VL comprises a CDR-L1 set forth in SEQ ID NO: 531, a CDR-L2 set forth in SEQ ID NO: 532, a CDR-L3 set forth in SEQ ID NO: 533, and wherein the VH comprises a CDR-H1 set forth in SEQ ID NO: 534, a CDR-H2 set forth in SEQ ID NO: 535, and a CDR-H3 set forth in SEQ ID NO: 536.

56. The activatable antibody of claim 53, wherein the VH comprises an amino acid sequence of SEQ ID NO: 602, or an amino acid sequence with at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 602; and the VL comprises an amino acid sequence of SEQ ID NO: 603, or an amino acid sequence with at least 90%, at least 95%, or at least 98% identity to SEQ ID NO: 603.

57. The activatable antibody of any one of claims 53-56, wherein the cleavable peptide is a substrate for a protease enriched in the pancreas of a subject.

58. The activatable antibody of claim 57, wherein the protease is trypsin.244FH13277368.1Attorney Docket No: TGY-0012559. The activatable antibody of claim 57, wherein the cleavable peptide is selected from SEQ ID NO: 192-233, or 448.

60. The activatable antibody of one of claims 53-59, wherein the masking peptide comprises an amino acid sequence which binds to one or more CDRs of the VH or VL region.

61. The activatable antibody of any one of claims 53-59, wherein the masking peptide is an antiidiotype antibody or an antigen binding fragment thereof.

62. The activatable antibody of any of the preceding claims, wherein the masking peptide does not bind to one or more CDRs of the activatable antibody.

63. The activatable antibody of any of the preceding claims, wherein the masking peptide inhibits antigen binding through steric hindrance, spatial occlusion, epitope adjacency, conformational restriction, or combinations thereof.

64. The activatable antibody of one of the preceding claims comprising two or more masking peptides.

65. The activatable antibody of claim 64, wherein the first masking peptide and the second masking peptide comprise leucine zipper coiled-coils.

66. The activatable antibody of claim 64, wherein the first masking peptide and the second masking peptide form parallel coiled-coils with high inter-coil affinities to reversibly impair antibody binding.

67. The activatable antibody of any one of claims 64-66, wherein the first masking peptide is linked to the VH domain and the second masking peptide is linked to the VL domain.

68. The activatable antibody of any one of claims 64-66, wherein the at least one masking peptide linked to the cleavable peptide and wherein the cleavable peptide is linked directly to the VL domain.

69. The activatable antibody of any one of claims 64-66, wherein the at least one masking peptide is linked to the cleavable peptide and wherein the cleavable peptide is linked directly to the VH domain.245FH13277368.1Attorney Docket No: TGY-0012570. The activatable antibody of any one of the preceding claims, wherein the at least one masking peptide is set forth in SEQ ID NO: 447.

71. The activatable antibody of any one of claims 53-69, wherein the at least one masking peptide is set forth in SEQ ID NO: 449.

72. The activatable antibody of any one of claims 53-71, wherein the at least one masking peptide inhibits binding of anti-CD3 to CD3.

73. The activatable antibody of any one of the preceding claims, wherein the masking peptide is less than 20 amino acids, less than 15 amino acids, less than 10 amino acids or less than 5 amino acids.

74. The activatable antibody of any one of the preceding claims, wherein the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VH domain.

75. The activatable antibody of any one of the preceding claims, wherein the masking peptide is linked directly to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VH domain.

76. The activatable antibody of any one of the preceding claims, wherein the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked directly to the VH domain.

77. The activatable antibody of any one of the preceding claims, wherein the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VL domain.

78. The activatable antibody of any one of the preceding claims, wherein the masking peptide is linked directly to the cleavable peptide and wherein the cleavable peptide is linked via a linker peptide to the VL domain.

79. The activatable antibody of any one of the preceding claims, wherein the masking peptide is linked via a linker peptide to the cleavable peptide and wherein the cleavable peptide is linked directly to the VL domain.246FH13277368.1Attorney Docket No: TGY-0012580. The activatable antibody of any one of the preceding claims, comprising a linker peptide linking the VH or VL with the cleavable peptide or linking the masking peptide with the cleavable peptide.

81. The activatable antibody of any one of the preceding claims, wherein the linker peptide is comprised of about 1 to 30 amino acids.

82. The activatable antibody of any one of the preceding claims, wherein the linker peptide is comprised of less than 30 amino acids, less than 20 amino acids, or less than 10 amino acids.

83. The activatable antibody of any one of the preceding claims, wherein the linker peptide is a GlySer linker.

84. The activatable antibody of any one of the preceding claims, wherein the linker peptide is comprised of an amino acid sequence of SEQ ID NOs: 436-438.

85. The activatable antibody of any one of the preceding claims, wherein the antibody is a humanized antibody, a chimeric antibody, or a human antibody.

86. The activatable antibody of claim 53, wherein the cleavable peptide is a substrate for ADAMTS-5.

87. The activatable antibody of any one of the preceding claims that inhibits IFNy, TNFa, or IL-6 or a combination thereof in vivo relative to a control antibody lacking the at least one masking peptide.

88. The activatable antibody of any one of the preceding claims that does not induce T cell proliferation in the absence of the protease and / or induces T cell proliferation in the presence of the protease.

89. The activatable antibody of any one of the preceding claims, wherein the T cells are CD4+ and / or CD8+ T cells.

90. A nucleic acid encoding the activatable antibody of any one of the preceding claims.247FH13277368.1Attorney Docket No: TGY-0012591. A vector comprising the nucleic acid of claim 90.

92. A host cell comprising the nucleic acid of claim 90 or 91.

93. A method of producing an activatable antibody, comprising culturing the host cell of claim 92 under conditions that produce the activatable antibody.

94. A pharmaceutical composition comprising the activatable antibody of any one of the preceding claims, and a pharmaceutically acceptable carrier.

95. A kit comprising the activatable antibody of any one of the preceding claims, and instructions for use.

96. A method of treating an autoimmune or inflammatory disease in a subject, comprising administering to the subject an activatable antibody of any one of the preceding claims or a pharmaceutical composition comprising the activatable antibody of claim 94.

97. The method of claim 79, wherein the activatable antibody or pharmaceutical composition is administered to the subject intravenously, intramuscularly, systemically, or locally.

98. Use of the activatable antibody of any one of the preceding claims or pharmaceutical composition of claim 94 in the manufacture of a medicament for the treatment of an autoimmune or inflammatory disease.

99. Use of the activatable antibody of any one of the preceding claims or pharmaceutical composition of claim 94 for the treatment of an autoimmune or inflammatory disease in a subject.248FH13277368.1

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