Activatable antibodies and methods of use thereof

1. An activatable antibody comprising an antigen-binding domain (ABD), wherein the ABD comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the N-terminus of the VH is fused to a first polypeptide shield moiety (S1), and the N-terminus of the VL is fused to a second polypeptide shield moiety (S2), wherein each of S1 and S2 is a polypeptide no more than about 50 amino acids long,

wherein association of S1 with S2 blocks binding of the ABD to its target,

wherein the ABD does not specifically bind to S1, S2, or association thereof,

wherein S1 comprises a first association moiety (B), and S2 comprises a second association moiety (C), wherein B and C are conjugated to each other by an isopeptide bond, and wherein S1 comprises the amino acid sequence of any one of SEQ ID NOs: 140, 142 and 227, and wherein S2 comprises the amino acid sequence of any one of SEQ ID NOs: 141 and 143; or wherein S1 comprises the amino acid sequence of any one of SEQ ID NOs: 141 and 143, and wherein S2 comprises the amino acid sequence of any one of SEQ ID NOs: 140, 142 and 227.

2. The activatable antibody of claim 1 , wherein B and C are conjugated to each other by a transglutaminase.

3. The activatable antibody of claim 1 , wherein the antibody is a bispecific antibody.

4. The activatable antibody of claim 1 , wherein the antibody is a Fab, a (Fab)2, an Fv, or a full-length antibody.

5. The activatable antibody of claim 1 , wherein the antibody is an antibody-drug conjugate (ADC).

6. The activatable antibody of claim 1 , wherein the ABD specifically binds to EGFR, CTLA-4, PD-1, CD-71, PD-L1, HER2, CD3, a-4 integrin, a-V integrin, a-4-b-1 integrin, a-4-b-7 integrin, AGR2, Anti-Lewis_Y, Apelin J receptor, APRIL, B7-H3, B7-H4, BCMA, BTLA, C5 complement, C-242, CA9, CA19-9, Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, IL27, IL27R, IL29, IL31, IL31R, Insulin Receptor, Jagged-1, Jagged-2, KIR, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, CD22, CD24, CD25, CD27, CD28, CD30, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117, MCSP, Mesothelin, MRP4, MUC1, Mucin-16, Na/K ATPase, Neutrophil elastase, NGF, Nicastrin, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NOV, OSM-R, CD125, CD132, CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACM5, CEACAM6, CLAUDIN-3, CLAUDIN-4, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTFG, CXCL10, CXCL13, CXCR1, CXCR2, OX-40L, OX-40R, PAR2, PDGF-AA, PDGF-BB, PDGFRa, PDGFRb, PD-1, PD-L1, PD-L2, Phospahtidyl-serine, P1GF, PSCA, PSMA, RAAG12, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EGFR, EGFRVIII, Endothelin b receptor (ETBR), ENPP3, EPCAM, EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF-8, FGFR-1, FGFR-2, FGFR-3, FGFR-4, Folate receptor, RAGE, SLC44A4, TNFa, STEAP1, STEAP2, TAG-72, TAPA1, TGFb, TIGIT, TIM3, TLR2, TLR4, TLR6, TLR7, TLR8, GAL3STI, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb/IIIa receptor, Gp130, GPNIVIB, GRP78, HER-2/neu, HGF, hGH, HVEM, Hyaluronidase, ICOS, INFa, INFb, INFg, IgE, TLR9, TMEM31, Sphingosine 1, Phosphate, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin Receptor, TRK-A, TRK B, uPAR, VCAM-1, VEGF, VEGF-A, IgE Receptor, IGF, IGF1R, IL1B, IL1R, IL2, IL2R, IL4, IL4R, IL6, IL6R, IL11, IL12, IL12p40, IL12R, IL12Rbl, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, or WISP-3.

7. A pharmaceutical composition comprising the activatable antibody of claim 1 and a pharmaceutically acceptable carrier.

8. The activatable antibody of claim 1 , wherein S1 and/or S2 further comprises a glycosylation peptide sequence.

9. The activatable antibody of claim 8 , wherein the glycosylation peptide sequence comprises the amino acid sequence of SEQ ID NO: 18.