Pro-antibody that reduces off-target toxicity

1 . An activatable antibody (aAb) comprising, in order, the following structure:

a first light chain comprising:

a first variable light region and constant light region;

a first heavy chain consisting of:

a first variable heavy region, a constant heavy region, and an Fc region; and

a cleavable linker between the constant light region and the first variable heavy region, wherein the cleavable linker prevents or reduces the first variable heavy region from pairing with the first variable light region to form a first antigen binding site against a first antigen; and

wherein cleavage of the cleavable linker releases the first variable heavy region to pair with the first variable light region to form the first antigen binding site to bind a first antigen.

2 . The aAb of claim 1 , wherein the aAb further comprises a second antigen binding site formed by a second variable light chain and a second variable heavy chain connected to the first heavy chain that binds a second antigen, and optionally a flexible non-cleavable linker between the second variable light chain and the second variable heavy chain.

3 . The aAb of claim 1 , wherein the first light chain, the first heavy chain, or both, further comprise an Fc region, a wild-type Fc region, a mutated Fc region, a monomeric wild type Fc region, a monomeric mutant Fc region, a dimeric wild type Fc region, or a dimeric mutant Fc region, a second variable heavy region and a second Fc region, or a second variable heavy region and a second Fc region and an uncleavable flexible linker and a second variable light region and a second heavy variable region, or a second Fc region and an uncleavable flexible linker and a cytokine.

4 . The aAb of claim 2 , wherein the first and second antigen are at least one of:

the same antigen; the first and second antigen are different; or the first and second antigen is the same antigen but the first antigen binding site and the second antigen binding site bind different epitopes of the same antigen;

the first antigen binding site or the second antigen binding site binds a tumor target;

the first antigen is a tissue specific surface antigen selected from ICAM1; VCAM1; EpCAM; extra domain B of fibronectin; melanoma-associated chondroitin sulfate proteoglycan (MCSP); melanoma-associated proteoglycan (MAPG); high molecular weight melanoma associated antigen (HMV-MAA); prostate specific membrane antigen (PSMA); epidermal growth factor receptor (EGFR); hepatocyte growth factor receptor (HGFR); fibroblast activation protein (FAP); carcinoembryonic antigen (CEA); cell-adhesion molecule (CAM); human B-cell maturation target (BCMA); placental growth factor (PLGF); folate receptor, insulin-like growth factor receptor (ILGFR); CD133; CD40; CD37; CD33; CD30; CD28; CD24; CD23; CD22; CD21; CD20; CD19; CD13; CD10; HER3; HER2; nonmuscle myosin heavy chain type A (nmMHCA); transferrin; epithelial cell adhesion molecule (EpCAM); annexin A 1; nucleotin, tenascin, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2; (VEGFR-2); aminopeptidase N, tie-1, tie-2, or c-Met;

the first antigen is selected from a protein, a portion of a protein, or a peptides encoded by at least one gene selected from: ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1; B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1; C19orfl0 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3 (MIP-la); CCL4 (MIP-lb); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-IRB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR 13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8 (CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wapl/Cipl); CDKNIB (p27Kipl); CDKNIC; CDKN2A (pl6INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CXCLIO (IP-IO); CXCL11 (I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GR02); CXCL3 (GR03); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; CGRP; Clq; Clr; Cl; C4a; C4b; C2a; C2b; C3a; C3b; DAB2IP; DES; DKFZp451J0118; DNCL1; DPP4; E-selectin; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENOI; EN02; EN03; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); Factor VII; Factor IX; Factor V; Factor Vila; Factor X; Factor XII; Factor XIII; FADD; FasL; FASN; FCER1A; FCER2; Fc gamma receptor; FCGR3A; FGF; FGFI (aFGF); FGF10; FGFII; FGF12; FGF12B; FGFI 3; FGFI 4; FGF16; FGFI 7; FGFI 8; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRTI (fibronectin); FLTI; FOS; FOSLI (FRA-1); FY (DARC); GABRP (GABAa); GAGEBI; GAGECI; GALNAC4S-6ST; GATA3; GDF5; GFII; GGTI; GMCSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (CIO); GRP; GSN (Gelsolin); GSTP1; glycoprotein (GP) Ilb/Illa; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; Her2; HGF; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); L-selectin; LAMA 5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB 1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSSI; MUCI (mucin); MYC; MYD88; NCK2; neurocan; NKG2D; NFKB1; NFKB2; NGF; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NRII2; NRII3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PARTI; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGE2; PGF; PGR; phosphacan; PIAS2; PIK3CG; plasminogen activator; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKDI; PRL; PROC; Protein C; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RAGE; RARB; RGSI; RGS13; RGS3; RNFIIO (ZNF144); ROB02; SI00A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Sprl); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; substance P; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB111; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-α; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSFIO (TRAIL); TNFSF11 (TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEML); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase lia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; thrombomodulin; thrombin; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-lb); XCR1 (GPR5/CCXCR1); YY1; and ZFPM2;

the first antigen binding site or the second antigen binding site binds a T-cell activator;

the T-cell activator is selected from CD3, 41BB or OX40;

the tumor target is selected from a tumor targeting antigen, HER1, HER2, HER3, GD2, carcinoembryonic antigens (CEAs), epidermal growth factor receptor active mutant (EGFRVIII), CD133, Fibroblast Activation Protein Alpha (FAP), Epithelial cell adhesion molecular (Epcam), Glypican 3 (GPC3), EPH Receptor A4 (EphA), tyrosine-protein kinase Met (cMET), IL-13Ra2, microsomal epoxide hydrolase (mEH), MAGE, Mesothelin, MUC16, MUC1, prostate stem cell antigen (PSCA), Wilms tumor-1 (WT-1), or a Claudin family protein;

the first antigen binding site or the second antigen binding site binds a T-cell marker; or

the T-cell marker is selected from CTLA-4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, or TIM-3.

5 . The aAb of claim 1 , wherein the cleavable linker is a protease cleavable linker.

6 . The aAb of claim 5 , wherein

the cleavable linker is cleaved by a tumor associated protease: MMP1, MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, uPA, FAPa, or Cathepsin B; or

the cleavable linker is cleaved by proteases upregulated during apoptosis or inflammation associated responses; or

the cleavable linker is cleaved by a caspase;

wherein the caspase is Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 11 or Caspase 12.

7 . The aAb of claim 1 , wherein the cleavable linker does not mask an antigen binding site.

8 . The aAb of claim 1 , further comprising:

an agent conjugated to the aAb; or

a cytokine attached to, or in a fusion protein with the aAb or an Fc region;

wherein the cytokine is selected from at least one of: growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; TNF-α; mullerian-inhibiting substance; gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors; platelet-growth factor; placental growth factor, transforming growth factors (TGFs); insulin-like growth factor-1 and-11; erythropoietin (EPO); osteoinductive factors; interferons; colony stimulating factors (CSFs); lymphotoxin-alpha; lymphotoxin-beta; CD27L; CD30L; FASL; 4-1 BBL; OX40L; TRAIL; IL-1; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-15; IL-18; IL-21; IL-22; IL-23; IL-33; IFN-a; IFN-b; IFN-g; IFN-g inducing factor (IGIF); bone morphogenetic protein (BMP); leukemia inhibitory factor (LIF); or kit ligand (KL).

9 . The aAb of claim 1 , wherein the aAb comprises an amino acid sequence selected from SEQ ID NOS: 1, 2, or 3.

10 . The aAb of claim 8 , wherein the agent is at least one of: a toxin or toxic fragment thereof; a microtubule inhibitor; a nucleic acid damaging agent; a detectable moiety; or a diagnostic agent.

11 . The aAb of claim 1 , further comprising a pharmaceutically acceptable diluent or carrier.

12 . An activatable antibody (aAb) comprising, in order, the following structure:

a first light chain comprising a first variable light region and a constant light region;

a cleavable linker; and

a first heavy chain comprising a first variable heavy region and a constant heavy region;

wherein the cleavable linker prevents or reduces the first variable heavy region from pairing with the first variable light region to form a first antigen binding site against a first antigen; and

wherein cleavage of the cleavable linker releases the first variable heavy region to pair with the first variable light region to form the first antigen binding site to bind a first antigen.

13 . The aAb of claim 12 , further comprising at least one of:

a first constant light region or a first constant heavy region, respectively; or

an Fc region attached to a first constant heavy region, wherein the Fc region is a wild-type or a mutant domain that modifies Fc receptor binding.

14 . The aAb of claim 12 , wherein the aAb further comprises a second antigen binding site formed by a second variable light chain and a second variable heavy chain connected to the first heavy chain that binds a second antigen, and optionally a flexible non-cleavable linker between the second variable light chain and the second variable heavy chain;

further comprises an Fc region that is a wild-type Fc region, a mutated Fc region, a monomeric wild type Fc region, a monomeric mutant Fc region, a dimeric wild type Fc region, or a dimeric mutant Fc region, a second variable heavy region and a second Fc region, or a second variable heavy region and a second Fc region and an uncleavable flexible linker and a second variable light region and a second heavy variable region, or a second Fc region and an uncleavable flexible linker and a cytokine;

further comprising a cytokine attached to, or in a fusion protein with the aAb or the Fc region, and wherein the cytokine is selected from at least one of: growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; TNF-alpha; mullerian-inhibiting substance; gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors; platelet-growth factor; placental growth factor, transforming growth factors (TGFs); insulin-like growth factor-1 and-11; erythropoietin (EPO); osteoinductive factors; interferons; colony stimulating factors (CSFs); lymphotoxin-alpha; lymphotoxin-beta; CD27L; CD30L; FASL; 4-1 BBL; OX40L; TRAIL; IL-1; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-15; IL-18; IL-21; IL-22; IL-23; IL-33; IFN-a; IFN-beta; IFN-gamma; IFN-gamma inducing factor (IGIF); bone morphogenetic protein (BMP); leukemia inhibitory factor (LIF); or kit ligand (KL).

15 . The aAb of claim 12 , wherein the first antigen is a tissue specific surface antigen selected from ICAM1; VCAM1; EpCAM; extra domain B of fibronectin; melanoma-associated chondroitin sulfate proteoglycan (MCSP); melanoma-associated proteoglycan (MAPG); high molecular weight melanoma associated antigen (HMV-MAA); prostate specific membrane antigen (PSMA); epidermal growth factor receptor (EGFR); hepatocyte growth factor receptor (HGFR); fibroblast activation protein (FAP); carcinoembryonic antigen (CEA); cell-adhesion molecule (CAM); human B-cell maturation target (BCMA); placental growth factor (PLGF); folate receptor, insulin-like growth factor receptor (ILGFR); CD133; CD40; CD37; CD33; CD30; CD28; CD24; CD23; CD22; CD21; CD20; CD19; CD13; CD10; HER3; HER2; nonmuscle myosin heavy chain type A (nmMHCA); transferrin; epithelial cell adhesion molecule (EpCAM); annexin A 1; nucleotin, tenascin, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2; (VEGFR-2); aminopeptidase N, tie-1, tie-2, or c-Met; or the first antigen is selected from a protein, a portion of a protein, or a peptides encoded by at least one gene selected from: ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1; C19orfl0 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3 (MIP-la); CCL4 (MIP-Ib); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-IRB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8 (CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wapl/Cipl); CDKNIB (p27Kipl); CDKNIC; CDKN2A (pl6INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CXCLIO (IP-IO); CXCL11 (I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GR02); CXCL3 (GR03); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; CGRP; Clq; CIR protein; CI; C4a; C4b; C2a; C2b; C3a; C3b; DAB2IP; DES; DKFZp451J0118; DNCL1; DPP4; E-selectin; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENOI; EN02; EN03; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); Factor VII; Factor IX; Factor V; Factor VIla; Factor X; Factor XII; Factor XIII; FADD; FasL; FASN; FCER1A; FCER2; Fc gamma receptor; FCGR3A; FGF; FGFI (aFGF); FGF10; FGFII; FGF12; FGF12B; FGFI 3; FGFI 4; FGF16; Fgfl7; Fgfl 8; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FUJ12584; FLJ25530; FLRTI (fibronectin); FLTI; FOS; FOSLI (FRA-1); FY (DARC); GABRP (GABAa); GAGEBI; GAGECI; GALNAC4S-6ST; GATA3; GDF5; GFII; GGTI; GMCSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (CIO); GRP; GSN (Gelsolin); GSTP1; glycoprotein IIb; glycoprotein Illa; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; Her2; HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMGB1; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-alpha; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFN-gamma; IFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL1A; IL1B; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; ILIA; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2; IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8RA; IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAKI; IRAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); L-selectin; LAMA 5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSSI; MUCI (mucin); MYC; MYD88; NCK2; neurocan; NKG2D; NFKB1; NFKB2; NGF; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NRII2; NRII3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PARTI; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGE2; PGF; PGR; phosphacan; PIAS2; PIK3CG; plasminogen activator; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKDI; PRL; PROC; Protein C; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RAGE; RARB; RGSI; RGS13; RGS3; RNFIIO (ZNF144); ROB02; SI00A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Sprl); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; substance P; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB111; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF-alpha; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSFIO (TRAIL); TNFSF11 (TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEML); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase lia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; thrombomodulin; thrombin; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-lb); XCR1 (GPR5/CCXCR1); YY1; and ZFPM2.

16 . The aAb of claim 12 , wherein the first antigen binding site binds a T-cell marker, or wherein the T-cell marker is selected from CTLA-4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, TIM-3, or wherein the first antigen binding site binds a T-cell activator, or wherein the T-cell activator is selected from CD3, 41BB or OX40.

17 . The aAb of claim 14 , wherein the second antigen binding site binds a T-cell marker, or wherein the T-cell marker is selected from CTLA-4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, TIM-3, or

wherein the second antigen binding site binds a T-cell activator, or wherein the T-cell activator is selected from CD3, 41BB or OX40.

18 . The aAb of claim 12 , wherein the cleavable linker is:

a protease cleavable linker; or

the cleavable linker is cleaved by a tumor associated protease: MMP1, MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, uPA, FAPa, or Cathepsin B; or

the cleavable linker is cleaved by proteases upregulated during apoptosis or inflammation associated responses; or

the cleavable linker is cleaved by a caspase; and the caspase is Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 11 or Caspase 12.

19 . The aAb of claim 12 , wherein the cleavable linker does not mask an antigen binding site.

20 . The aAb of claim 12 , further comprising an agent conjugated to the aAb, or wherein the agent is at least one of: a toxin or toxic fragment thereof; a microtubule inhibitor; a nucleic acid damaging agent; a detectable moiety; or a diagnostic agent.

21 . The aAb of claim 12 , wherein the aAb comprises an amino acid sequence selected from SEQ ID NOS: 1, 2, or 3.

22 . A nucleic acid that encodes an aAb, wherein the aAb encoded by the nucleic acid comprises, in order, the following structure:

a first light chain comprising:

a first variable light region and constant light region;

a first heavy chain consisting of:

a first variable heavy region, a constant heavy region, and an Fc region; and

a cleavable linker between the constant light region and the first variable heavy region;

wherein the cleavable linker prevents or reduces the first variable heavy region from pairing with the first variable light region to form a first antigen binding site against a first antigen; and

wherein cleavage of the cleavable linker releases the first variable heavy region to pair with the first variable light region to form the first antigen binding site to bind a first antigen.

23 . A cell that comprises a nucleic acid that encodes an aAb of claim 22 .

24 . An activatable Antibody (aAb), in order, comprising:

a first variable light region;

a cleavable linker;

a first variable heavy region; and

an Fc region;

wherein the cleavable linker prevents or reduces the first variable heavy region from pairing with the first variable light region to form a first antigen binding site against a first antigen, wherein the cleavable linker does not mask the antigen binding site; and

wherein cleavage of the cleavable linker releases the first variable heavy region to pair with the first variable light region to form the first antigen binding site to bind a first antigen.

25 . A cell that expresses an activatable antibody (aAb) comprising, in order, the following structure:

a first light chain comprising:

a first variable light region;

a cleavable linker; and

a first heavy chain comprising:

a first variable heavy region;

wherein the cleavable linker prevents or reduces the first variable heavy region from pairing with the first variable light region to form a first antigen binding site against a first antigen; and

wherein cleavage of the cleavable linker releases the first variable heavy region to pair with the first variable light region to form the first antigen binding site to bind a first antigen.

26 . The cell of claim 25 , wherein the cell is a T cell or a mesenchymal stem cell.

27 . The cell of claim 25 , wherein the aAb further comprises a transmembrane sequence that anchors the aAb to the surface of a T cell to form a chimeric antigen receptor, wherein the cell is a CAR T cell.

28 . An activatable antibody (aAb) comprising, in order, the following structure: a first light chain comprising:

a first variable light region;

a cleavable linker; and

a first heavy chain comprising:

a first variable heavy region;

wherein the cleavable linker prevents or reduces the first variable heavy region from pairing with the first variable light region to form a first antigen binding site against a first antigen;

wherein cleavage of the cleavable linker releases the first variable heavy region to pair with the first variable light region to form the first antigen binding site to bind a first antigen; and

wherein the aAb does not contain a masking moiety that blocks an antigen binding site.

29 . A nucleic acid that encodes an aAb, wherein the aAb encoded by the nucleic acid comprises, in order, the following structure:

a first light chain comprising:

a first variable light region;

a cleavable linker; and

a first heavy chain comprising:

a first variable heavy region;

wherein the cleavable linker prevents or reduces the first variable heavy region from pairing with the first variable light region to form a first antigen binding site against a first antigen;

wherein cleavage of the cleavable linker releases the first variable heavy region to pair with the first variable light region to form the first antigen binding site to bind a first antigen; and

wherein the aAb does not contain a masking moiety that blocks an antigen binding site.

30 . A cell that comprises a nucleic acid that encodes an aAb of claim 29 .

31 . The aAb of claim 14 ,

wherein the first and second antigen are at least one of:

the same antigen; the first and second antigen are different; or the first and second antigen is the same antigen but the first antigen binding site and the second antigen binding site bind different epitopes of the same antigen;

the first antigen binding site or the second antigen binding site binds a tumor target; or

the tumor target is selected from a tumor targeting antigen, HER1, HER2, HER3, GD2, carcinoembryonic antigens (CEAs), epidermal growth factor receptor active mutant (EGFRVIII), CD133, fibroblast Activation Protein Alpha (FAP), epithelial cell adhesion molecular (Epcam), glypican 3 (GPC3), EPH Receptor A4 (EphA), tyrosine-protein kinase Met (cMET), IL-13Ra2, microsomal epoxide hydrolase (mEH), MAGE, Mesothelin, MUC16, MUC1, prostate stem cell antigen (PSCA), Wilms tumor-1 (WT-1), or a Claudin family member.