Modified antibodies and immunoglobulin formats

Abstract

Provided herein are modified antibodies or antibody binding fragments thereof that have inhibited binding activity for their cognate antigens. The antibodies or antibody binding fragments comprise an antigen binding domain and two or more blocking elements that are operably linked through a cleavable linker to the antigen binding domain.

Description

[0001] MODIFIED ANTIBODIES AND IMMUNOGLOBULIN FORMATS

[0002]

[0001] The present application claims the benefit of U.S. Provisional Application No. 63 / 647,999, filed on May 15, 2024, U.S. Provisional Application No. 63 / 699,332. filed on September 26, 2024, U.S. Provisional Application No. 63 / 738.027, filed on December 23. 2024. U.S. Provisional Application No. 63 / 753,145, filed on February 3, 2025, and U.S. Provisional Application No. 63 / 782,772, filed on April 3, 2025, the entire contents of each of which are incorporated herein by reference.

[0003] 1. BACKGROUND

[0004]

[0002] Immunotherapy has great potential in cancer treatment, but many of the hopes for advantages of immunotherapy have not yet been realized. Cancer immunotherapy approaches involve manipulating the activity of a subject’s immune system to eliminate cancer cells while limiting immune-related adverse events that might harm healthy cells. Modulating immune responses using agonistic antibodies to activate co-stimulatory molecules on immune system cells has been suggested as a possible tactic for cancer therapy.

[0005]

[0003] In particular, T-cell engagers transiently tether T cells to tumor cells and mediate T cell-directed tumor killing. However, adoption of T-cell engagers in solid tumors is limited by the scarcity of tumor antigens with sufficient differential expression between tumor and normal tissue. T-cell engagers that are preferentially active in the tumor microenvironment may enable the safe targeting of more solid tumor antigens 3

[0006]

[0004] As such, there is an unmet need here is a need for antibody therapeutics for improved targeting and delivery, in particular for use in diagnosis and treatment of cancers.

[0007] 2. SUMMARY

[0008]

[0005] Tire disclosure relates to modified antibodies or antibody binding fragments thereof (collectively referred to as modified ABs herein) that have inhibited binding activity for their cognate antigens. Tire modified ABs comprise at least a first antigen binding site (e.g., a VH / VL), and at least one and preferably two or more blocking elements that are operably linked through a cleavable linker (e.g., containing a disease-specific protease cleavage site) to the antigen binding site. The blocking elements, by being operably linked to the antigen binding site, can inhibit binding to the cognate antigen for example, by binding to the antigen binding site or stcrically blocking the antigen binding site. The inventors have discovered, as described and exemplified herein, that operably linking two blocking elements to an antigen binding site surprisingly provides better inhibition of binding to the cognate antigen than a single blocking element.

[0009]

[0006] Provided herein are prodrug polypeptide compositions that comprise (i) a first binding domain (BD1); (ii) a first protease cleavable linker peptide (LP); (iii) a first activity inhibitory peptide (AIP1) (e.g., a blocking element) having an affinity for BD1 and a second activity inhibitory peptide (AIP2) (e.g., a blocking element) having an affinity for BD 1. Tire combination of AIP 1 and AIP2 can provide synergistic inhibitory activity to BD 1. The prodrug polypeptide composition can further comprise an optional half-life extension domain (XD) (e.g., a half-life extension element).

[0010]

[0007] Tire disclosure also relates to a prodrug polypeptide composition, comprising: (a) a first target antigen binding domain (BD1), (b) a second target antigen binding domain (BD2), (c) a first activity inhibitory peptide (AIP1) having an affinity for BD1 and a second activity inhibitory peptide (AIP2) having an affinity for BD1, (d) a first protease cleavable linker peptide (LP), and an optional half-life extension domain (XD).

[0011]

[0008] The AIP can comprise an avidity (av) for BD1 (AIM-avBDl) greater than sum of the affinities for BD1 of the first and second activity inhibitory peptides (AIP1- avBDl + AIP2- avBDl).

[0012]

[0009] The first blocking element (BD1) and the second binding domain (BD2) can be independently capable of binding to an antigen or a cell surface antigen, or to a plurality of antigens or cell surface antigens. The first blocking element (BD1) and the second binding domain (BD2) can be independently capable of binding to a cell surface antigen associated with a cancer cell, a tumor, or a tumor-associated microenvironment, or to a plurality of antigens or cell surface antigens.

[0013]

[0010] Tire first binding domain (BD1) is capable of binding to any desired antigen or a cell surface antigen. For example, the first binding domain (BD1) is capable of binding to a cell surface antigen associated with a cancer cell, a tumor, or a tumor-associated microenvironment. In particular, the first binding domain (BD1) is capable of binding to a polypeptide selected from CD19, CD22. CD30, CD33. CD79b, HER2, trop2, nectin4, tissue factor, and folate receptor alpha.

[0014] [OH] The first binding domain (BD1) is capable of binding to a polypeptide selected from a tumor antigen selected from EGFR, PSMA, EpCAM, BCMA, 5T4, KLK2, AFP, Axl, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD38. CD44, CD52, CD56. CD70, CD71, CD74, CD79b, CEACAM5, c-MET DLL3, EphA2, FAP, FGFR2. FGFR3, glypican-3, FLT-3. FOLR1, gpNMB, HER2. HPV-16 E6, HPV-16 E7. ITGA3. SLC39A6, Mesothelin, MucL Mucl6, NaPi2b, Nectin-4, P-cadherin, Prolactin R, PSCA, PTK7, RORL SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, and WT1.

[0015]

[0012] Tire first binding domain (BD1) is capable of binding to a polypeptide selected from a tumor antigen selected from an immune modulatory protein selected from CTLA-4, CD27, CD 137, 2B4, TIGIT, CD155, ICOS. HVEM, CD40L, LIGHT. TIM-1. 0X40, DNAM-1, PD-L1. PD1, PD-L2, CD8, CD40. CEACAM1, CD48, CD70. A2AR, CD39, CD73. B7-H3, B7-H4, BTLA. IDO1, IDO2. TDO, KIR. LAG-3, TIM-3, VISTA, IL6-R, IL-6, TNFa, CD 19, CD20, CD22, CD52, integrin a4, integnn a4b7, CD1 la, CTLA4-Ig fhsion, IL-17, IL12 / 23, IL12, IL23, TGF-beta, IL-2, IL-21, IL-18, IL2RA, IL2RB, IL2RG, IL21R, IL18R1, IL18RAP, IL12RB1, IL12RB2, CD4, 4-1BB, 4-1BBL, GITR, GITRL and a claudin, such as claudin 1, claudin 4, claudin 6, claudin 7 or claudin 18.2.

[0016]

[0013] For example, the first binding domain (BD1) is capable of binding to a cell surface antigen associated with an autoimmune disease, immune disorder, or condition, if desired.

[0017]

[0014] The prodrug polypeptide can be formulated as a pharmaceutical formulation in an amount effective to treat or reduce the severity of an autoimmune disease selected from asthma, Systemic Lupus Erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis (RA), irritable inflammatory bowel disease (IBD), type 1 diabetes, or graft-versus-host-disease (GvHD).

[0018]

[0015] The first binding domain BD 1 can comprise an antibody or antibody binding fragment that was generated against the polypeptide sequence of the first activity inhibitory peptide (AIP1) or (AIP2) the first activity inhibitory peptide or both, or b) AIP1 or AIP2 or both is not the native epitope for the antibody or fragment.

[0019]

[0016] Tire protease cleavable linker peptide (LP) (e.g., protease cleavable linker) can be capable of being selectively cleaved in a tumor microenvironment in a human subject. The protease cleavable linker peptide (LP) can be capable of being selectively cleaved in an inflammatory microenvironment in a human subject.

[0020]

[0017] The protease cleavable linker can comprise an amino acid sequence X1-X2-X3-X4-X5-X6-X7- X8-X9-X10-X11-X12 (Formula L) wherein XI is selected from X2, X3, X4, X5, X6, X7, X8, X9, X10, Xll and X12 are each independently any amino acid or one, two, three or four of XI, X2, X3, X4, X5, X6, X7, X8, X9. X10, Xll and X12 is absent, and wherein LP is recognized by at least one protease (i) selected from the group consisting of neutrophil elastase, proteinase 3, and cathepsin G. kallikrein, a thrombin, a chymase, a carboxypeptidase, a cathepsin, an elastase, a PR-3, a granzyme, a calpain. a matrix metalloproteinase (MMP), a fibroblast activation protein (FAP), an ADAM metalloproteinase, a plasminogen activator, a caspase, a tryptasc. and a tumor cell surface protease and / or (ii) present in a human tumor microenvironment and / or (iii) present in a human inflammatory microenvironment, and wherein the LP is not significantly cleaved by at least one protease selected from the group consisting of Factor Xa, thrombin, and hepsin.

[0021]

[0018] The protease cleavable linker peptide (LP) has a greater cleavage susceptibility in a tumor microenvironment or an inflammatory microenvironment than a reference microenvironment. The reference microenvironment can be selected from human serum and a normal primary cell population. The protease cleavable linker peptide (LP) can have a greater cleavage susceptibility to a tumor- associated protease and / or an inflammation-associated protease than a reference protease. The protease cleavable linker peptide (LP) can have a greater cleavage susceptibility to a tumor-associated protease and / or an inflammation-associated protease than a reference protease selected from a serum protease.

[0019] The protease cleavable linker peptide (LP) can be cleaved at a greater rate in a tumor microenvironment or an inflammatory environment in a human subject than in the systemic circulation or in a physiologically normal tissue of the human subject. The protease cleavable linker can be cleaved at a greater rate in a disease microenvironment in a human subject than in physiologically normal tissue of the human subject.

[0022]

[0020] The protease cleavable linker peptide (LP) is generally not significantly cleaved in an in vitro cell culture environment comprising a mammalian producer cell line.

[0023]

[0021] When present, the half-life extension domain (XD) (e g., half-life extension element) can be human serum albumin or a variant or fragment thereof (HSA) or an anti-HSA antibody or fragment thereof. Tire half-life extension domain (XD) can sterically block an activity of the first binding domain (BD1).

[0024]

[0022] The activity inhibitory peptide (AIP) (e.g., blocking element) can comprises (i) a first activity inhibitory peptide (AIP1) from about 7 amino acids in length to about 13 amino acids in length, (ii) a second activity inhibitory peptide (AIP2) from about 7 amino acids in length to about 13 amino acids in length, and (iii) a peptide linker operably linking (i) and (ii).

[0025]

[0023] Tire first activity inhibitory peptide (AIP1) and the activity inhibitory peptide (AIP2) can comprise the amino acid sequence of SEQ ID NO: 496 or SEQ ID NO: 497. The first activity inhibitory peptide (AIP1) or the second activity inhibitory peptide (AIP2) or the combination thereof can be from about 5 to about 15 amino acids in length. The first activity inhibitory peptide one (A1P1) or second activity inhibitory peptide (AIP2) or the combination thereof can be from about 7 to about 13 amino acids in length. The first activity inhibitory peptide one (AIP1) or second activity inhibitory peptide (AIP2) can comprise the same amino acid sequence.

[0026]

[0024] Tire first activity inhibitory peptide one (AIP1) or second activity inhibitory peptide (AIP2) can be derived from a naturally occurring polypeptide. The first activity inhibitory peptide one (AIP1) or second activity- inhibitory peptide (AIP2) can be non-naturally occurring peptides. Hie first activity inhibitory peptide one (AIP1) or second activity inhibitory- peptide (AIP2) can comprise non-canonical amino acids.

[0027]

[0025] The first activity inhibitory peptide one (AIP1) or second activity inhibitory peptide (AIP2) can be operably linked by a non-cleavable linker.

[0028]

[0026] The prodrug polypeptide composition can further comprise a pharmaceutical agent selected from a small molecule, a radionuclide, a peptide, a polypeptide, a nucleic acid, or a cell therapy.

[0029]

[0027] The prodrug polypeptide composition can be formulated as a pharmaceutical formulation suitable for intravenous administration to a human subject in need thereof. The prodrug polypeptide composition can be formulated as a pharmaceutical formulation suitable for intravenous administration to a human subject having cancer or an autoimmune disease, disorder or condition.

[0030]

[0028] The disclosure also relates to a prodrug polypeptide composition, comprising: (a) a first target antigen binding domain (BD1), (b) a second target antigen binding domain (BD2), (c) a first activity- inhibitory peptide (AIP 1 ) having an affinity for BD 1 and a second activity inhibitory peptide (AIP2) having an affinity for BD1, wherein AIM comprises an inhibitory activity (inh) for BD1 (AIM- inh BD1) greater than sum of the inhibitory activity for BD 1 of the first and second activity inhibitory peptides (AIP1- inhBDl + AIP2- inhBDl), (d) a first protease cleavable linker peptide (LP), and (e) an optional half-life extension domain (XD).

[0031]

[0029] Provided herein is an isolated multi-specific antibody comprising: (a) a first polypeptide complex comprising at least a first and a second polypeptide sequence, wherein the first polypeptide sequence comprises P1-LP1 -BD1L (Formula I), and wherein the second polypeptide sequence comprises P1-LP1- BD1H-BD1CH (Formula II), and (b) a second polypeptide complex comprising at least a third and a fourth polypeptide sequence, wherein the first polypeptide sequence comprises P2-LP2-BD2L (Fonnula III), and wherein the second polypeptide sequence comprises P2-LP2-BD2H-BD2CH (Formula IV). BD1L and BD1H form BD1, comprising a CD3 binding domain. BD2L and BD2H form BD2, comprising a target antigen binding domain. LP1 and LP2 independently comprise a protease cleavable linker peptide. Pl comprises a first activity inhibitory peptide (AIP1) having an affinity for BD1. P2 comprises a second activity inhibitory peptide (AIP2) having an affinity for BD2. The first polypeptide sequence can comprise P1-XD-LP1-BD1L (Formula la). XD comprises a half-life extension domain. The second polypeptide sequence can comprises? 1-XD-LP1-BD1H-BD1 CH (Formula Ila). XD comprises a half-life extension domain. The third polypeptide sequence can comprise P2-XD-LP2-BD2L (Formula Illa). XD comprises a half-life extension domain. The fourth polypeptide sequence comprises P2-XD- LP2-BD2H-BD2CH (Formula IVa). XD comprises a half-life extension domain.

[0032]

[0030] Provided herein is isolated multi-specific antibody comprising, in any order: BD 1 comprising a CD3 binding domain; BD2 comprising a target antigen binding domain; LI and L2 independently comprising a linker peptide; LP comprising a protease cleavable linker peptide; Pl comprising a first activity inhibitory peptide (AIP1) having an affinity for BD1; P2 comprising a second activity inhibitory peptide (AIP2) having an affinity for BD1; optionally, P3 comprising a third activity inhibitory peptide (AIP3) having an affinity for BD2; and optionally. P4 comprising a fourth activity inhibitory peptide (AIP4) having an affinity for BD2.

[0033]

[0031] This disclosure also related to T-cell engagers that comprise (i) a first antigen binding domain that has binding specificity to an antigen on a T-cell, (ii) a target antigen binding domain that has binding specificity to a target antigen, (iii) a first blocking element and a second blocking element each having binding specificity for the first antigen binding domain, (iv) a first protease cleavable linker that operably links tire first blocking element to the first antigen binding domain and a second protease cleavable linker that operably links the second blocking element to the first antigen binding domain; and (v) an optional half-life extension element.

[0034]

[0032] Tire antigen on the T-cell can be CD3 epsilon, CD3 gamma, CD3 delta, CD 19, CD20, CD45, CD28, CS2, CD3, CD4, LFA1, CD40, CD52, CD19, CD22, CD80, CD86, CD267, CD269, CD268, or CS32B. Preferably, first antigen binding domain has binding specificity to CD3 epsilon.

[0035]

[0033] The target antigen can be an antigen associated with a cancer cell, a tumor, or a tumor-associated microenvironment.

[0036]

[0034] The target antigen can be EGFR, PSMA, EpCAM, BCMA, 5T4, KLK2, AFP, Axl, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37. CD38, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, CEACAM5, c-MET, DLL3, EphA2, FAP, FGFR2, FGFR3, glypican-3. FLT-3, FOLR1, gpNMB, HER2, HPV-16 E6. HPV-16 E7, ITGA3, SLC39A6, Mesothelin, Mucl. Mucl6. NaPi2b, Nectin-4, P-cadherin, Prolactin R. PSCA, PTK7, R0R1. SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, or WT1 .

[0035] The target antigen can be an immune modulatory protein selected from CTLA-4, CD27, CD 137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, 0X40, DNAM-1, PD-L1, PD1, PD-L2, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, VISTA, IL6-R, IL-6, TNFa, CD 19, CD20, CD22, CD52, integrin a4, integrin a4b7, CDlla, CTLA4-Ig fusion, IL-17, IL12 / 23, IL12, IL23, TGF-beta, IL-2, IL-21, IL-18, IL2RA, IL2RB, IL2RG, IL21R, IL18R1, IL18RAP, IL12RBL IL12RB2, CD4, 4- IBB, 4-1 BBL. GITR, GITRL and a claudin, such as claudin 1, claudin 4, claudin 6, claudin 7 or claudin 18.2.

[0037]

[0036] Preferably, the target antigen is EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1.

[0037] Tire first antigen binding domain and target antigen binding domain can each comprise an antibody or antigen binding fragment thereof. For example, the first antigen binding domain can be a single domain antibody, a Fab or a single-chain variable fragment (scFv). The target antigen binding domain can be a single domain antibody, Fab or scFv.

[0038]

[0038] In some T-cell engager formats, the first antigen binding domain can be a scFv and the second antigen binding domain is a Fab. In some T-cell engager formats, the first antigen binding domain can be a Fab and the second antigen binding domain is a scFv.

[0039]

[0039] Typically, the first antigen binding domain and tire target antigen binding domain are operably linked. The first antigen binding domain and the target antigen binding domain can be operably linked through a linker. The one or more linkers can be a synthetic linker, a peptide linker, or a chemical bond. Preferably, the one or more linkers is a peptide linker. Tire peptide linker can be a short peptide linker having about 4 amino acids to about 6 amino acids. The peptide linker can be a long peptide linker having about 5 amino acids to about 10 amino acids.

[0040]

[0040] The T-cell engager formats can comprise a first protease cleavable linker and a second protease cleavable linker that can each independently comprise a sequence that is capable of being cleaved by a protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A. an elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), a fibroblast activation protein (FAP), an ADAM metalloproteinase, a plasminogen activator, a cathepsin, a caspase, a tr ptase. and a tumor cell surface protease. Tire first protease cleavable linker and the second protease cleavable linker can each independently comprise a sequence that is cleaved by cathepsin selected from tire group consisting of cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K. cathepsin L, and cathepsin G. The first protease cleavable linker and the second protease cleavable linker can each independently comprise a sequence that is cleaved by a matrix metalloprotease (MMP) selected from the group consisting ofMMPl, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, and MMP14.

[0041]

[0041] The first protease cleavable linker and tire second protease cleavable linker can be cleaved by different proteases. The first protease cleavable linker and the second protease cleavable linker are cleaved by the same proteases.

[0042]

[0042] The T-cell engager formats comprise a blocking element that can be a peptide from about 5 amino acids in length to about 17 amino acids in length, and the second blocking element can be a peptide from about 5 amino acids in length to about 17 amino acids in length. The first blocking element can be a peptide from about 7 amino acids in length to about 13 amino acids in length, and the second blocking element can be a peptide from about 7 amino acids in length to about 13 amino acids in length. Tire first blocking element can be a peptide from about 5 amino acids in length to about 15 amino acids in length, and the second blocking element can be a peptide from about 5 amino acids in length to about 15 amino acids in length.

[0043]

[0043] The T-cell engager formats preferably comprise two or more blocking elements. For example, a first blocking element and second blocking element, which can comprise the same amino acid sequence. The first blocking element and the second blocking element can comprise different amino acid sequences. The first blocking element and the second blocking element can each comprise an epitope for the first antigen binding domain. The first blocking element and the second blocking element can comprise the same or different epitopes.

[0044]

[0044] The first blocking element and the second blocking element can be derived from a naturally occurring polypeptide or protein. The first blocking element and the second blocking element can be non- naturally occurring peptides. Tire first blocking element and the second blocking element can comprise non-canonical amino acids.

[0045]

[0045] Typically, the optional half-life extension element is present. The half-life extension element can comprises a serum albumin or a serum albumin peptide, an binding domain that binds serum albumin (e.g,. antibody or antigen antigen-binding fragment thereof, such as an scFV or sdAb), transferrin or a transferrin peptide, an binding domain that binds transferrin (e.g,. antibody or antigen antigen-binding fragment thereof, such as an scFV or sdAb), an immunoglobulin Fc or fragment thereof and the like. Typically, the half-life extension element is an antigen binding polypeptide which binds human serum albumin, such as a single domain antibody fragment (sdAb). The sdAb can compete with neonatal Fc receptor (FcRn) for binding to HSA. The sdAb can have binding specificity for domain one of human serum albumin. The sdAb can be a heavy chain variable domain (VH), a variable heavy domain of heavy chain (VHH), a single domain shark variable domain of new antigen receptor (VNAR), or a light chain variable (VL) domain.

[0046]

[0046] Tire half-life extension element can be an immunoglobulin Fc. The immunoglobulin Fc can comprise knob-in-hole heterodimerization modification

[0047]

[0047] The T-cell engagers disclosed herein can further comprise a third protease cleavable linker that operably links the half-life extension element to the T-cell engager. The third protease cleavable linker can operably link the half-life extension element to the C-terminus of the T-cell engager. The third protease cleavable linker can operably links the half-life extension element to the N-terminus of the T-cell engager.

[0048] Tire T-cell engager can further comprise a blocking element having binding specificity for the target antigen binding domain. The blocking element can comprise a target antigen protein or fragment thereof. For example, the blocking element can comprise an antigen-binding fragment selected from a single domain antibody, a Fab, or a scFv.

[0048]

[0049] The T-cell engager can further comprise a second target antigen binding domain having specificity for a target antigen. The T-cell engager can further comprise a third target antigen binding domain having specificity for a target antigen. Tire second and third target antigen binding domains can have binding specificity for a tumor antigen, such as a tumor antigen selected from EGFR, PSMA, EpCAM, BCMA, 5T4, KLK2, AFR Axl, B7-H3, Cadherm-6, CAIX, CD117, CD123. CD138. CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD38, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, CEACAM5, c-MET, DLL3, EphA2, FAP, FGFR2, FGFR3, glypican-3, FLT-3, FOLR1, gpNMB, HER2, HPV-16 E6, HPV-16 E7, ITGA3, SLC39A6, Mesothelin, Mucl, Mucl6, NaPi2b, Nectin- 4, P-cadherin, Prolactin R. PSCA, PTK7, R0R1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, and WT1. Tire second and third target antigen binding domain preferably binds EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1.

[0049]

[0050] The T-cell engager can further comprise an antigen binding domain having specificity to a costimulatory signaling molecule. Tire co-stimulatory molecule can be CD28, 4- IBB, ICOS, CD27, CD2, CD40L, GITR, 0X40 and CD226.

[0050]

[0051] The T-cell engager can further comprise an antigen binding domain having specificity for an immune checkpoint inhibitor. The immune checkpoint inhibitor can be 2B4. TIGIT, CD155. ICObi, HVEM. LIGHT. DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD40, CEACAM1. CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDOL IDO2, TDO, KIR, LAG-3, TIM-3, and VISTA.

[0052] The T-cell engager can comprise two polypeptide chains.

[0051]

[0053] Exemplary T-cell engager can comprise (a) a first antigen binding domain comprising an antibody Fab or Fv that has binding specificity for an antigen on a T-cell; (b) a target antigen binding domain comprising an antibody Fab or Fv that has binding specificity for a target antigen; (c) a first blocking element and a second blocking element each having binding specificity for the first antigen binding domain; (d) a first protease cleavable linker that operably links the first blocking element to the first antigen binding domain and a second protease cleavable linker that operably links the second blocking element to the first antigen binding domain; and (e) an optional half-life extension element. The T-cell engager can comprise a first polypeptide comprising: (i) a first heavy chain Fab fragment (VH + CHI) or a first heavy chain variable domain (VH); (ii) a second heavy chain Fab fragment (VH + CHI) or a second heavy chain variable domain (VH); (iii) a first blocking element having specificity for the first antigen binding domain; and (iv) a first protease cleavable linker that operably links the first blocking element to the first antigen binding domain or target antigen binding domain; (b) a second polypeptide comprising: (i) a first light chain Fab fragment (VL + CL) or light chain variable domain (VL), that is complementary to the first heavy chain Fab fragment (VH + CHI) or a first heavy chain variable domain (VH) of the first polypeptide; (ii) tire second light chain Fab fragment (VL + CL), or light chain variable domain (VL), that is complementary to second heavy chain Fab fragment (VH + CHI) or a second heavy chain variable domain (VH) of the second polypeptide, (iii) a second blocking element having specificity for the first antigen binding domain; (iv) a second protease cleavable linker that operably links the second blocking element to the first antigen binding domain.

[0052]

[0054] Such exemplary T-cell engagers can further comprise a half-life extension element. Tire half life extension element can be serum albumin or a serum albumin peptide or an antibody or antigen-binding fragment thereof (e.g.. scFv or sdAb) that binds serum albumin. The half life extension element can be an scFv or sdAb that binds serum albumin and the first polypeptide and / or the second polypeptide comprise the scFv or sdAb. When the T-cell engager comprises two polypeptides either of both polypeptide can further comprise tire half-life extension element. For example, the first polypeptide can further comprise the scFv or sdAb. Tire first polypeptide further can comprise an Fc region and tire scFv or sdAb can be linked to the Fc region. The first polypeptide can comprise a first heavy chain antibody Fab fragment (VH + CHI) and a second heavy chain variable domain (VH). The second polypeptide can comprise a first light chain antibody Fab fragment (VL + CL) that is complimentary to the VH + CHI on the first polypeptide and a second VH that is complimentary to the VL on the second polypeptide, which form two binding sites. The first polypeptide can comprise a first heavy chain variable domain (VH) and a second heavy chain antibody Fab fragment (VH + CHI). The second polypeptide can comprise a first light chain variable domain (VL) that is complimentary' to the first VH on the first polypeptide and a second light chain antibody Fab fragment (VL + CL) that is complimentary to the second VH + CHI, which form two binding sites.

[0053]

[0055] The T-cell engagers disclosed herein can comprise a structure shown in any one of FIGs. 3A-3F, 17A-17P, and 18A-18L, 19A-19I, 20A-20I, 29A-29C, and 39A-34B.

[0054]

[0056] In certain exemplary T-cell engagers, the first antigen binding domain can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 999, a CDR2 having the amino acid sequence of SEQ ID NO: 1000, and a CDR3 having the amino acid of SEQ ID NO: 1001, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1002, a CDR2 having the amino acid sequence of SEQ ID NO: 1003, and a CDR3 having the amino acid of SEQ ID NO: 1004. The first antigen binding domain can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 995 and a light chain variable region having the amino acid sequence of SEQ ID NO: 996.

[0055]

[0057] In certain exemplary T-cell engagers, the first antigen binding domain can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1005, a CDR2 having the amino acid sequence of SEQ ID NO: 1006. and a CDR3 having the amino acid of SEQ ID NO: 1007, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1008, a CDR2 having the amino acid sequence of SEQ ID NO: 1009, and a CDR3 having the amino acid of SEQ ID NO: 1010.

[0056]

[0058] In certain exemplary T-cell engagers, the first antigen binding domain can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 997 and a light chain variable region having the amino acid sequence of SEQ ID NO: 998.

[0057]

[0059] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 959, a CDR2 having the amino acid sequence of SEQ ID NO: 960, and a CDR3 having the amino acid of SEQ ID NO: 961, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 962, a CDR2 having the amino acid sequence of SEQ ID NO: 963, and a CDR3 having the amino acid of SEQ ID NO: 964.

[0060] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 947 and a light chain variable region having the amino acid sequence of SEQ ID NO: 948.

[0058]

[0061] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 965, a CDR2 having the amino acid sequence of SEQ ID NO: 966, and a CDR3 having the amino acid of SEQ ID NO: 967, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 968, a CDR2 having the amino acid sequence of SEQ ID NO: 969, and a CDR3 having the amino acid of SEQ ID NO: 970.

[0059]

[0062] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 949 and a light chain variable region having the amino acid sequence of SEQ ID NO: 950.

[0060]

[0063] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 977, a CDR2 having the amino acid sequence of SEQ ID NO: 978, and a CDR3 having the amino acid of SEQ ID NO: 979, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 980, a CDR2 having the amino acid sequence of SEQ ID NO: 981, and a CDR3 having the amino acid of SEQ ID NO: 982.

[0061]

[0064] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 953 and a light chain variable region having the amino acid sequence of SEQ ID NO: 954.

[0062]

[0065] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 983, a CDR2 having the amino acid sequence of SEQ ID NO: 984, and a CDR3 having the amino acid of SEQ ID NO: 985, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 986, a CDR2 having the amino acid sequence of SEQ ID NO: 987, and a CDR3 having the amino acid of SEQ ID NO: 988.

[0063]

[0066] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 955 and a light chain variable region having the amino acid sequence of SEQ ID NO: 956.

[0067] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 989, a CDR2 having the amino acid sequence of SEQ ID NO: 990, and a CDR3 having the amino acid of SEQ ID NO: 991, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 992, a CDR2 having the amino acid sequence of SEQ ID NO: 993, and a CDR3 having the amino acid of SEQ ID NO: 994.

[0064]

[0068] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 957 and a light chain variable region having the amino acid sequence of SEQ ID NO: 958.

[0065]

[0069] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 971, a CDR2 having the amino acid sequence of SEQ ID NO: 972, and a CDR3 having the amino acid of SEQ ID NO: 973, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 974, a CDR2 having the amino acid sequence of SEQ ID NO: 975, and a CDR3 having the amino acid of SEQ ID NO: 976.

[0066]

[0070] In certain exemplary T-cell engagers, the target antigen binding domain can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 951 and a light chain variable region having the amino acid sequence of SEQ ID NO: 952.

[0067]

[0071] In certain exemplary T-cell engagers, the first heavy chain variable region and tire second heavy chain variable region can be operably linked with a peptide comprising an amino acid sequence of SEQ ID NO: 920, SEQ ID NO: 921, SEQ ID NO: 923, SEQ ID NO: 924. The first light chain variable region and tire first heavy chain variable region can be operably linked with a peptide comprising an amino acid sequence of SEQ ID NO: 920. SEQ ID NO: 924, SEQ ID NO: 926, or SEQ ID NO: 927.

[0068]

[0072] In certain exemplary T-cell engagers, the first blocking element and the second blocking element can comprise the amino acid sequence of SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 496, and SEQ ID NO: 497, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 496, and SEQ ID NO: 497.

[0069]

[0073] In certain exemplary’ T-cell engagers, the protease cleavable linker comprises the amino acid sequence selected from SEQ ID NOs: 510 to 535. 707, 708. 942, 943. 944, 945. 946, or 1011.

[0074] In certain exemplary T-cell engagers, the half-life extension element can be a sdAb that has binding specificity for human serum albumin comprising a CDR1, CDR2 and CDR3; wherein CDR1 comprises any one of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 37, or a variant of any of tire foregoing comprising up to about 3 amino acid substitutions; CDR2 comprises any one of SEQ ID NO: 2, SEQ ID NO: 9. SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 38, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; and CDR3 comprises any one of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 12 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions

[0070]

[0075] In certain exemplary T-cell engagers, the sdAb can comprise: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2. or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. or a variant thereof comprising up to about 3 amino acid substitutions; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7. or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; (13) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; or (14) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions.

[0071]

[0076] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 716; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 824.

[0072]

[0077] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 716; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 825.

[0073]

[0078] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 717; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 826.

[0074]

[0079] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 717; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 825.

[0075]

[0080] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 719; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0076]

[0081] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 721; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0077]

[0082] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 723; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0078]

[0083] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 725; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 466.

[0079]

[0084] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 727; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 828.

[0085] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 729; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 830.

[0080]

[0086] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 731; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 832.

[0081]

[0087] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 734; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 835.

[0082]

[0088] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 734; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 836.

[0083]

[0089] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 735; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 835.

[0084]

[0090] The T-cell engager can comprise first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 735; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 836.

[0085]

[0091] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 737; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0086]

[0092] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 739; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0087]

[0093] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 741; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0088]

[0094] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 743; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0095] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 748; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 837.

[0089]

[0096] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 750; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 837.

[0090]

[0097] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 753; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0091]

[0098] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 755; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0092]

[0099] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 757; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0093]

[0100] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 759; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0094]

[0101] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 764; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 841.

[0095]

[0102] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 764; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 842.

[0096]

[0103] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 766; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 841.

[0097]

[0104] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 775; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 845.

[0105] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 775; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 846.

[0098]

[0106] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 777; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 845.

[0099]

[0107] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 777; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 846.

[0100]

[0108] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 777; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0101]

[0109] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 779; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0102] [HO] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 782; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0103] [Hl] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 784; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697.

[0104]

[0112] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 788; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 849.

[0105]

[0113] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 788; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 850.

[0106]

[0114] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 792; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 849.

[0115] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 792; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 850.

[0107]

[0116] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 799; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 852.

[0108]

[0117] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 801; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 854.

[0109]

[0118] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 803; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 856.

[0110]

[0119] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 809; and) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 859.

[0111]

[0120] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 809; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 860.

[0112]

[0121] The T-cell engager can comprise first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 811; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 859.

[0113]

[0122] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 811: and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 860.

[0114]

[0123] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 815; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 861.

[0115]

[0124] The T-cell engager can comprise a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 816; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 861.

[0125] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 819; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 861.

[0116]

[0126] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 820; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 861.

[0117]

[0127] The T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 821; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0118]

[0128] Tire T-cell engager can comprise a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 822; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 862.

[0119]

[0129] The T-cell engager can comprise: (a) a first antigen binding domain that has binding specificity for CD3 comprising a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 999, a CDR2 having tire amino acid sequence of SEQ ID NO: 1000, and a CDR3 having tire amino acid of SEQ ID NO: 1001 and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1002. a CDR2 having tire amino acid sequence of SEQ ID NO: 1003, and a CDR3 having the amino acid of SEQ ID NO: 1004 or a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1005, a CDR2 having the amino acid sequence of SEQ ID NO: 1006, and a CDR3 having the amino acid of SEQ ID NO: 1007, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1008, a CDR2 having the amino acid sequence of SEQ ID NO: 1009. and a CDR3 having the amino acid of SEQ ID NO: 1010;

[0120]

[0130] (b) a target antigen binding domain comprising a heavy chain variable region comprising a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 959. a CDR2 having the amino acid sequence of SEQ ID NO: 960, and a CDR3 having the amino acid of SEQ ID NO: 961, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 962, a CDR2 having the amino acid sequence of SEQ ID NO: 963, and a CDR3 having the amino acid of SEQ ID NO: 964; a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 965. a CDR2 having tire amino acid sequence of SEQ ID NO: 966, and a CDR3 having the amino acid of SEQ ID NO: 967, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 968, a CDR2 having the amino acid sequence of SEQ ID NO: 969, and a CDR3 having the amino acid of SEQ ID NO: 970; a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 977, a CDR2 having the amino acid sequence of SEQ ID NO: 978, and a CDR3 having tire amino acid of SEQ ID NO: 979, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 980, a CDR2 having the amino acid sequence of SEQ ID NO: 981, and a CDR3 having the amino acid of SEQ ID NO: 982; a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 983, a CDR2 having the amino acid sequence of SEQ ID NO: 984, and a CDR3 having the amino acid of SEQ ID NO: 985, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 986, a CDR2 having the amino acid sequence of SEQ ID NO: 987, and a CDR3 having the amino acid of SEQ ID NO: 988; or a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 971, a CDR2 having the amino acid sequence of SEQ ID NO: 972, and a CDR3 having the amino acid of SEQ ID NO: 973, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 974, a CDR2 having the amino acid sequence of SEQ ID NO: 975, and a CDR3 having the amino acid of SEQ ID NO: 976;

[0121]

[0131] (c) a first blocking element and a second blocking element each having binding specificity for the first antigen binding domain; wherein the first and second blocking element comprise the amino acid sequence of SEQ ID NO: 489. SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492. SEQ ID NO: 493, SEQ ID NO: 496, or SEQ ID NO: 497.

[0122]

[0132] (d) a first protease cleavable linker that operably links the first blocking element to the first antigen binding domain and a second protease cleavable linker that operably links the second blocking element to tire first antigen binding domain, wherein the protease cleavable linker comprises an amino acid sequence selected from SEQ ID NOs: 510 to 535, 707, 708, 942, 943, 944, 945, 946, or 1011; and

[0123]

[0133] (e) an optional half-life extension element; wherein the half-life extension element comprises a CDR1, CDR2 and CDR3; wherein CDR1 comprises any one of SEQ ID NO: 1. SEQ ID NO: 4. SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 37, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; CDR2 comprises any one of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 38, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; and CDR3 comprises any one of SEQ ID NO: 3, SEQ ID NO: 6. SEQ ID NO: 12 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions.

[0124]

[0134] In such exemplary T-cell engagers, the first antigen binding domain and the target antigen binding domain, can each independently comprise VH and VL or VH + CHI and VL + CL.

[0135] In such exemplary T-cell engagers, the first antigen binding domain and the target domain can be operably linked through a linker having an amino acid sequence of SEQ ID NO: 920, SEQ ID NO: 921, SEQ ID NO: 923, SEQ ID NO: 924 SEQ ID NO: 926, or SEQ ID NO: 927, and combinations thereof.

[0125]

[0136] In such exemplary T-cell engagers, the VH or VH + CHI of the first antigen binding domain and the VH or VH + CHI of the target antigen binding domain can be operably linked through a linker having the amino acid sequence of SEQ ID NO: 920, SEQ ID NO: 921. SEQ ID NO: 923, or SEQ ID NO: 924.

[0126]

[0137] In such exemplary T-cell engagers, the VL or VL + CL of the first antigen binding domain and the VL + CL of the target antigen binding domain are operably linked through a linker having the amino acid sequence of SEQ ID NO: 920, SEQ ID NO: 924, SEQ ID NO: 926, or SEQ ID NO: 927.

[0127]

[0138] Additional exemplary T-cell engagers can comprise a first polypeptide and a second polypeptide. The first polypeptide comprising (i) a first heavy chain Fab fragment (VH + CHI); (ii) a target antigen binding domain comprising an scFV that bind target antigen; and (iii) a first blocking element having specificity for the first antigen binding domain; and (iv) a first protease cleavable linker that operably links the first blocking element to the first antigen binding domain or the scFV that binds target antigen. The second polypeptide can comprising: (i) a first light chain Fab fragment (VL + CL); (iii) a second blocking element having specificity for tire first antigen binding domain; and (iv) a second protease cleavable linker that operably links the second blocking element to the first antigen binding domain.

[0128]

[0139] Such exemplary T-cell engager can further comprise a half life extension element. The half life extension element can be serum albumin or a serum albumin peptide or an scFv or sdAb that binds serum albumin. The half life extension element can be an scFv or sdAb that binds serum albumin and the first polypeptide and / or the second polypeptide comprise the scFv or sdAb that binds serum albumin.

[0129]

[0140] The first polypeptide can further comprises tire scFv or sdAb. Tire scFv or sdAb is operably linked to CHI of the first heavy chain Fab fragment or operably linked to the scFV that binds target antigen. Tire scFv or scFv or sdAb can be operably linked to CHI of tire first heavy chain Fab fragment or operably linked to the scFV that binds target antigen through a linker that is optionally protease cleavable.

[0130]

[0141] The T-cell engager can comprises a structure shown in any one of FIGs. 17J or 17L.

[0131]

[0142] Also disclosed herein are nucleic acid encoding the T-cell engagers. The nucleic acid can be RNA. The nucleic acid can be mRNA or circular RNA (circRNA). Provided herein is a vector that can comprise the nucleic acids encoding the T-cell engager. The vector can be an adeno-associated virus (AAV), retroviral vector, lentiviral vector, or adenoviral vector. A host cell can comprise the nucleic acid or vector.

[0143] The disclosure further relates to pharmaceutical compositions comprising the T-cell engager, the nucleic acid, the vector, or host cell.

[0132]

[0144] Also provided herein are A methods treating a cancer, comprising administering to a subject in need

[0133] 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0134]

[0145] The drawings are not necessarily to scale or exhaustive. Instead, the emphasis is generally placed upon illustrating the principles of the inventions described herein. The accompanying drawings, which constitute a part of the specification, illustrate several embodiments consistent with the disclosure and, together with the description, serve to explain the principles of tire disclosure. In the drawings:

[0135]

[0146] FIGs. 1A-1Z and 1ZA-1ZK are schematics illustrations depicting various modified antibodies and antibody binding fragments with peptide blockers.

[0136]

[0147] FIGs. 2A-2R are graphs showing the activity of conditionally active CD3 agonist prodrugs in a primary human PBMC assay. These graphs demonstrate the activity of either cleaved (open diamond) or intact (closed diamond) conditionally active CD3 agonist prodrugs as compared to an unblocked, positive control molecule (WW50871 / WW50870; open square), using either proliferation (FIGs. 2A, 2C, 2E, 2G, 21, 2K, 2M, 20, and 2Q) or IFNgamma (FIGs. 2B, 2D, 2F, 2H, 2J, 2L, 2N, 2P, and 2R) production as a readout for T cell activation. EC50 values for each protein are shown below their respective graphs. The results confirm that the intact T cell engager molecules have inducible activity in primary human PBMCs.

[0137]

[0148] FIGs. 3A-3F are schematic illustrations depicting conditionally active T-cell engager prodrugs.

[0138]

[0149] FIGs. 4A-4H are images of SDS-PAGE gels showing anti-human serum albumin VHH antibodies derived from llama. The VHHs were 6xHis-tagged (SEQ ID NO: 917) (FIGs. 4A-4C). Fc- tagged (FIGs. 4D-4G), or 6xHis- (SEQ ID NO: 917) and AviTagged (FIG. 4H). NR refers to nonreduced conditions, and R refers to reduced conditions.

[0139]

[0150] FIGs. 5A-5F are tables showing sequence alignments of humanized variants of llama anti -human serum albumin VHH antibodies. Alignments of each llama VHH, the chosen human germline Vh template protein(s), and humanized variants are shown in FIG. 5A (1HUM19), FIG. 5B (3HUM181), FIG. 5C (2HUM45 on a IGHV3-23 backbone), FIG. 5D, (2HUM45 on an IGHV3-11 backbone). FIG. 5E (1HUM19 variants with modified CDR sequences), and FIG. 5F (3HUM181 variants with modified CDR sequences). The CDRs are indicated. FIG. 5A shows SEQ ID NOs. 928-930 and 39-47, respectively, in order of appearance. FIG. B shows SEQ ID NOs. 931-933 and 56-65, respectively, in order of appearance. FIG. 5C shows SEQ ID NOs. 69, 934, 930 and 66-70, respectively, in order of appearance. FIG. 5D shows SEQ ID NOs. 78, 935, 936 and 71-79, respectively, in order of appearance. FIG. 5E shows SEQ ID NOS 937-939, 48-55 and 426-728, respectively, in order of appearance. FIG. 5F shows SEQ ID NOs 940-941, 930 and 429-430, respectively, in order of appearance.

[0140]

[0151] FIGs. 6A-6J are images of SDS-PAGE gels showing humanized anti-human serum albumin VHH antibodies. The humanized VHH antibodies were run under non-reduced conditions (FIGs. 6A-6J) and reduced conditions (FIGs. 6F-6J).

[0141]

[0152] FIGs. 7A and 7B are graphs showing predicted pharmacokinetics (PK) (pg / mL) overtime of anti-albumin VHHs with varying binding affinities to mouse (FIG. 7A) or human serum albumin (FIG. 7B).

[0142]

[0153] FIGs. 8A-8G are graphs showing that an anti-CD3 blocking element prevents tire induction of cytokine release syndrome (CRS) in N-terminal hCD3a mice. C57BL / 6 mice in which the N-terminus of CD3E was humanized (genOway) were injected with a single 10 ug dose of either unmasked or a bivalent anti-human CD3 antibody with two blocking elements administered intravenously (2 mice per group). Blood was collected at baseline, 2 hours and 6 hours post antibody administration. Serum was analyzed by Luminex Mouse Discovery assay at all collected time points to quantify tire amount of TNFa, IFNy, IL-4, IL-6, CCL2, CXCL10, and CCL4.

[0143]

[0154] FIGs. 9A-9G are graphs showing that an anti-CD3 blocking element prevents tire induction of CRS by bivalent anti-CD3 antibodies in Pan hCD3 mice. C57BL / 6 mice where the entire CD3E, 5, and y subunits was humanized (genOway) were injected with a single 10 ug dose of either unmasked or bivalent anti -human CD3 antibody with two blocking elements administered IV (2 mice per antibody). Blood was collected at baseline, 2 hours and 6 hours post antibody administration. Serum was analyzed by Luminex Mouse Discovery assay at all collected time points to quantify the amount of TNFa. IFNy, IL-4. IL-6. CCL2. CXCL10, and CCL4.

[0144]

[0155] FIGs. 10A-10D are graphs showing that anti-CD3 blocking element prevents the induction of CRS by anti-CD3 / anti-CD19 TCE in CD34+humanized NSG mice bearing CD19+Raji tumors. Four- week-old female NSG mice were humanized with human CD34+hematopoietic stem cells. Twelve weeks post engraftment, Hum -NSG mice were subcutaneously injected with CD19+Raji tumor cells on the right flank and allowed to grow for 2 weeks. Hum -NSG mice were administered a single dose of either unmasked anti-CD3 / anti-CD19 TCE (20 pg), TCE with a single anti-CD3 blocking element (200 pg), or TCE with two anti-CD3 blocking elements (200 pg). Two mice per group were bled at the 2-hour and 24- hour timepoints, and two mice for the 6-hour and 48-hour timepoints. Serum was analyzed by Luminex Human Performance assay at all collected time points to quantify TNFa, IFNy, IL-6, and CCL4.

[0145]

[0156] FIGs. 11A-11D show that anti-CD3 / anti-CD19 POC T-cell engager (TCE or as described herein as a modified Ab) is cleaved by dissociated human tumor cells and can activate patient T-cells. A primary human non-small cell lung cancer sample was dissociated into a single cell suspension, then incubated with CD 19+ Raji cells and various T cell engager molecules before activation of the primary human tumor infiltrating T cells was assessed as a measurement of activity. Cleavage-dependent activation of primary, tumor infiltrating CD4+ (FIG. HA) CD8+ (FIG. 11B) T cells following incubation with inducible, T cell engager molecule containing two anti-CD3 blocking elements. The increase in T cell activation was significant for both populations, relative to the non-cleavable control containing two anti- CD3 blocking elements (FIGS. 11C and HD). These data demonstrate that primary human tumor samples can remove the two anti-CD3 blocking elements and induce activity in a cleavage-dependent manner.

[0146]

[0157] FIGs 12A-12D are representative graphs depicting cytotoxicity assay data for TCEs targeting CD 19 or EGFR. FIG. 12A is a graph showing the activity of unmasked anti-CD3 / anti-CD19 TCE (WW51310 / WW50870) or half-life extended anti-CD3 / anti-CD19 TCE with two anti-CD3 blocking elements and anti-CD19 blocking element (WW51425 / WW51083) in the flow cytometry-based cytotoxicity assay. FIG. 12B is a graph showing the activity of unmasked anti-CD3 / anti-CD19 TCE (WW51310 / WW50870) or half-life extended anti-CD3 / anti-CD19 TCE with two anti-CD3 blocking elements and anti-CD19 blocking element (WW51425 / WW51083) in the Raji HiBiT cytotoxicity assay. Both the flow cytometry -based cytotoxicity assay and the Raji HiBiT cytotoxicity assay s quantify the T cell -mediated killing of CD 19+ Raji target cells induced by TCEs. While the intact form of

[0147] WW5 1425 / WW51083 is not active in either assay, protein that has been cleaved in vitro to remove the anti-CD3 blocking element and the anti-CD19 blocking element demonstrates activity that is comparable to the unmasked control (WW51310 / WW50870) in both assays. FIG. 12C is a graph showing the activity of various EGFR-targeting TCEs in the SKOV3 HiBiT assay. FIG. 12D is a graph showing the activity of various EGFR-targeting TCEs in the HT-1376 LDH cytotoxicity assay. Both SKOV and HT-1376 cells express EGFR and are the target cells for which cytotoxicity is monitored after treatment with TCEs. Unmasked anti-CD3 / anti-EGFR TCE (WW51625 / WW50870) is active in both assays. Addition of a C- terminal half-life extension domain does not affect activity, as observed for WW51758 / WW50870 in the SKOV3 HiBiT assay. However, inclusion of two anti-CD3 blocking elements (WW51759 / WW51083) results in decreased cytotoxicity. Blocking of both anti-CD3 and the anti-EGFR arms further attenuates activity, as observed for intact WW51773 / WW 1083 in the HT-1376 LDH assay; however, in vitro cleavage and removal of all masking domains restores the activity of WW51773 / WW51083. The activity of related anti-CD3 / anti-CD19 and anti-CD3 / anti-EGFR TCEs is indicated in Tables 21-24.

[0148]

[0158] FIGs. 13A-13C are graphs showing that the anti-CD3 blocking element can be used to inhibit the activity of antibodies that bind the N-terminus of human CD3E. including SP34. SP34 antibodies were expressed with either no blocking element, a single anti-CD3 blocking element on the heavy chain or light chain, or anti-CD3 blocking elements on both chains. Antibody activity was assessed by quantifying the proliferation of PBMCs induced by engaging CD3. When a single anti-CD3 blocking element is present on the heavy chain, the intact antibody (WW51413 / WW51416) is less active than the SP34 antibody control (WW51415 / WW51416) with no blocking element. However, removal of the blocking element by in vitro cleavage restores tire activity ofWW51413 / WW51416 to levels observed forthe control with no blocking element (FIG. 13A). Similarly, a single anti-CD3 blocking element on the light chain inhibits the activity of SP34 (WW51415 / WW51414), but once removed, does not impact the antibody’s ability to induce proliferation (FIG. 13B). The activity of tire SP34 antibody with anti-CD3 peptide masks on both chains (WW51413 / WW51414) was further attenuated relative to antibodies with a single peptide (FIG. 13C). These data indicate that the anti-CD3 peptide mask is an effective blocking moiety that can be used to block antibodies with similar linear epitopes of human CD3F.

[0149]

[0159] FIG. 14A is a table showing the percentage of cleavage activity in human dissociated tumors (DTs) and human normal primary cell and serum stability cleavable linkers having SEQ ID NO: 513, SEQ ID NO: 707, and SEQ ID NO: 708. Each of the linkers demonstrated high levels of cleavage by human dissociated tumors (DTs) and were stable when incubated with normal human primary cells. Linkers were stable when incubated with human serum for 72 hours and when incubated with high concentrations of proteases associated with healthy serum and tissues.

[0150]

[0160] FIG. 14B is a graph showing the average percent activity after each inducible IL-2 prodrug was incubated with dissociated tumor samples was calculated and averaged for each linker across four time points. The data demonstrate that linkers are cleaved at variable rates, with linkers having SEQ ID NO: 707 and SEQ ID NO: 708 having faster average cleavage rates than linker having SEQ ID NO: 513.

[0151]

[0161] FIG. 14C shows the percentage activity after each inducible IL-2 prodrug was incubated with dissociated tumor samples was calculated and grouped by tumor type: bladder cancers, gastric cancers, renal cell carcinoma (RCC), colorectal cancer (CRC), non-small cell lung cancer (NSCLC), and gynecologic cancers. Each point represents data from an individual patient sample. The data demonstrates cleavage of linkers by patient samples.

[0152]

[0162] FIGs. 15A-15D are graphs showing cleavage of inducible IL-2 prodrugs with pan-cancer linkers (SEQ ID NO: 513, 707, and 708) by dissociated tumor samples. NSG female mice were inoculated with either a medium dose (2 million cells) or a high dose (5 million cells) of HT- 1376 or HCT116 cells. Tire medium dose groups were also humanized with PBMCs prior to tumor cell inoculation. Once established, tumors were excised from mice and dissociated to generate a single cell suspension. Dissociated tumor samples were then incubated with inducible IL-2 prodrug containing tumor protease-sensitive linkers, specifically SEQ ID NO: 513, SEQ ID NO: 707, or SEQ ID NO: 708. Data are graphed as percent activity of IL-2, indicative of cleavage of inducible IL-2 prodrugs by dissociated tumor, after 24 or 48 hours of incubation. FIGs. 15A and 15B shows data from HT-1376 dissociated tumors. FIGs. 15C and 15D show data from HCT116 dissociated tumors. These data demonstrate cleavage of linkers having SEQ ID NO: 513, SEQ ID NO: 707, and SEQ ID NO: 708 by HT-1376 and HCT116 tumor samples.

[0153]

[0163] FIGs. 16A and 16B are graphs showing pan-cancer linkers are cleaved by CDX dissociated tumors from various indications. Linker having SEQ ID NO: 513, 707, and 708 were cleaved by dissociated tumor samples from mice inoculated with HCT116, HT29, A549, LoVo, or SK-OV-3. Tire graphs show percent activity of IL-2, indicative of cleavage of inducible prodrugs by dissociated tumor incubated for 24 (FIG. 16A) or 48 hours (FIG. 16B).

[0154]

[0164] FIGs. 17A-17P are schematic illustrations depicting T-cell engager prodrugs described herein.

[0155]

[0165] FIGs. 18A-18H are schematic illustrations depicting T-cell engager prodrugs that comprises a blocking element having binding specificity for the target antigen binding domain described herein. The target antigen binding domain can be any desired target antigen, such as those listed in Table 1. An exemplary target antigen depicted in FIGs. 18A-18D is EpCAM. An exemplary target antigen depicted in FIGs. 18E-18H is B7H3.

[0156]

[0166] FIGs. 19A-19I are schematic illustrations depicting T-cell engagers that comprise an antigen binding domain having binding specificity for CD3, two or three target antigen binding domains having binding specificity for one or more tumor antigens, a half-life extension element, and a protease cleavable linker. Hie anti-tumor target antigen binding domain can be any desired target antigen, such as those listed in Table 1. The two or three anti -tumor target antigen binding domains can have binding specificity to the same tumor target antigen or different anti-tumor target antigens. The half-life extension element functions as a blocking element by sterically blocking the first antigen binding domain.

[0167] FIGs. 20A-20I are schematic illustrations depicting T-cell engager prodrugs that comprise an antigen binding domain having binding specificity for CD3, two or three target antigen binding domains having binding specificity for one or more tumor antigens, two anti-CD3 blocking elements, a half-life extension element, and a protease cleavable linker. The anti-tumor target antigen binding domain can bind to any desired target antigen, such as those listed in Table 1. Tire two or three anti -tumor target antigen binding domains can have binding specificity to the same tumor target antigen or different anti-tumor target antigens.

[0157]

[0168] FIGs. 21A-21D are graphs showing the activity of T-cell engager prodrags targeting CD3 and EGFR as percentage of cytotoxicity or percentage of killing. FIG. 21A is a graph showing the activity of C-terminal half-life extended anti-CD3 Fab / anti-EGFR scFv TCE (WW51758 / WW50870, anti-EGFR scFv in VH / VL orientation, circles) or C-tenninal half-life extended masked anti-CD3 Fab / anti-EGFR scFv TCE (WW51759 / WW51083, anti-EGFR scFv in VH / VL orientation, squares) in the SKOV-3 HiBit cytotoxicity assay. There is a 43x window of inducibility between intact and cleaved TCE. FIG. 21B is a graph showing the activity of pre-cleaved C-terminal half-life extended unmasked anti-CD3 / anti-EGFR DVD-Fab TCE (WW51934 / WW51935, circles) or C-terminal half-life extended masked anti-CD3 / anti- EGFR DVD-Fab TCE (WW51972 / WW51973, squares) in the HCT-116 (LDH) cytotoxicity assay. The intact molecule shows no killing activity while the cleaved molecule has an EC50 of 91pM. In FIG. 21C, C-terminal half-life extended anti-EGFR scFv (VH / VL orientation) with a masked anti-CD3 Fab is tested intact (squares) and pre-cleaved (circles) in the SKOV-3 HiBit cytotoxicity assay. There is a 17x window of inducibility between intact and cleaved TCE. In FIG. 21D, a masked anti-CD3 scFv (VL / VH orientation) with N-terminal half-life extended masked anti-EGFR Fab demonstrates no target cell killing, squares. A pre-cleaved version of the same TCE (circles) illustrates that removal of the anti-CD3 mask and half-life extension domain leads to a potent TCE in the HCT-116 (LDH) cytotoxicity assay with an EC50 of 71pM.

[0158]

[0169] FIGs. 22A-22D are graphs showing the activity of T-cell engager prodrags targeting CD3 and B7-H3 as percentage of cytotoxicity. FIG. 22A is a graph showing the activity of C-terminal half-life extended masked anti-CD3 Fab / anti-B7-H3 scFv TCE (WW52329 / WW51690, anti-B7-H3 scFv in VL / VH orientation, black circles) or C-tenninal half-life extended masked anti-CD3 Fab / anti-B7-H3 scFv TCE (WW52333 / WW51690, anti-B7-H3 scFv in VH / VL orientation, circles) in the HCT-116 LDH cytotoxicity assay. Neither TCE shows killing activity. FIG. 22B is a graph showing the activity- of precleaved C-terminal half-life extended unmasked anti-CD3 / anti-B7-H3 DVD-Fab TCE (WW51963 / WW51966, circles) or C-terminal half-life extended masked anti-CD3 / anti-B7-H3 DVD-Fab TCE (WW51986 / WW51989, squares) in the SKOV3 HiBiT cytotoxicity assay. Tire intact molecule shows no killing activity while the cleaved molecule has an EC50 of 4.1pM. In FIG. 22C, N-terminal half-life extended anti-B7-H3 scFv (VL / VH orientation) with a masked anti-CD3 Fab is tested intact (squares) and pre-cleaved (circles). The intact molecule shows no killing activity while the cleaved molecule has an EC50 of 4.8pM. In FIG. 22D, a masked anti-CD3 scFv (VL / VH orientation) with N- terminal half-life extended masked anti-B7-H3 Fab demonstrates low levels of target cell killing at high concentrations of TCE, squares. A pre-cleaved version of the same TCE (circles) illustrates that removal of the anti-CD3 mask and half-life extension domain leads to a highly potent TCE in the SKOV3 HiBiT cytotoxicity assay.

[0159]

[0170] FIGs. 23A-23C are graphs showing the activity of T-cell engager prodrugs targeting CD3 and EpCAM as percentage of cytotoxicity. FIG. 23A is a graph showing the activity of pre-cleaved (triangles) and intact (circles) C-terminal half-life extended anti-CD3 / anti-EpCAM DVD-Fab TCE (WW51975 / WW51976) in the SK-BR-3 HiBit cytotoxicity assay. The intact molecule shows no killing activity while the cleaved molecule has an EC50 of 39pM. FIG. 23B is a graph showing the activity of unmasked, non-half life extended anti-CD3 / anti -EpCAM DVD-Fab TCE (WW52245 / WW51937, diamonds), C-terminal half-life extended unmasked anti-CD3 / anti-EpCAM DVD-Fab TCE with an EpCAM blocking domain (WW52238 / WW51937, triangles) or C-terminal half-life extended masked anti-CD3 / anti-EpCAM DVD-Fab TCE with an EpCAM blocking domain (WW52241 / WW51975, circles) in the SK-BR-3 HiBit cytotoxicity assay. The intact molecule shows no killing activity while the unmasked non-half life extended TCE has an EC50 of 1.7pM and the unmasked anti-CD3 / anti-EpCAM DVD-Fab TCE with an EpCAM blocking domain demonstrates some blockade with an EC50 of 432pM. In FIG. 23C, the activity of unmasked, non-half life extended anti-EpCAM / anti-CD3 DVD-Fab TCE (WW52246 / WW52247. diamonds). C-terminal half-life extended unmasked anti-EpCAM / anti-CD3 DVD-Fab TCE with an EpCAM blocking domain (WW52240 / WW52247, triangles) or C-terminal halflife extended masked anti-EpCAM / anti-CD3 DVD-Fab TCE with an EpCAM blocking domain (WW52244 / WW52243, circles) in the SK-BR-3 HiBit cytotoxicity assay. The intact molecule shows no killing activity while the unmasked non-half life extended TCE has an EC50 of 7.3pM and tire unmasked anti-CD3 / anti-EGFR DVD-Fab TCE with an EpCAM blocking domain demonstrates some blockade with an EC50 of 40pM.

[0171] FIGs. 24A-24C are graphs showing the activity of T-cell engager prodrugs targeting CD3 and DLL-3 as percentage of cytotoxicity. FIG. 24A is a graph showing the activity of C-terminal half-life extended ummasked anti-CD3 Fab / anti-DLL-3 scFv TCE (WW51565 / WW50870, anti-DLL-3 scFv in VI I / VL orientation, circles) or C-terminal half-life extended unmasked anti-CD3 Fab / anti-DLL-3 scFv TCE (WW52566 / WW50870, anti-DLL-3 scFv in VL / VH orientation, squares) in the SHP-77 LDH cytotoxicity assay. Neither TCE shows killing activity. FIG. 24B is a graph showing the activity of precleaved and intact unmasked C-terminal half-life extended anti-CD3 / anti-DLL-3 DVD-Fab TCE with short / short linkers (WW51940 / WW51941, squares and circles, respectively) in the SHP-77 LDH cy totoxicity assay. There is a 1 Ox window between intact and cleaved suggesting there is a small amount of steric blockade from the anti-HSA domain. In FIG. 24C, the activity is shown for pre-cleaved and intact unmasked C-terminal half-life extended anti-CD3 / anti-DLL-3 DVD-Fab TCE with long / short linkers (WW51942 / WW51941, squares and circles, respectively) in the SHP-77 LDH cytotoxicity assay. There is a 5 Ox window between intact and cleaved suggesting there is a small amount of steric blockade from the anti-HSA domain.

[0160]

[0172] FIGs. 25A-25C are graphs showing the activity of T-cell engager prodrugs targeting CD3 and STEAP-1 as percentage of cytotoxicity. FIG. 25A is a graph showing the activity of intact (squares) and pre-cleaved (circles) C-tenninal half-life extended ummasked anti-CD3 Fab / anti-STEAP- 1 scFv TCE (WW51875 / WW50870. anti-STEAP-1 scFv in VL / VH orientation) in the LnCAP LDH cytotoxicity assay. Neither intact or cleaved TCE shows killing activity. FIG. 25B is a graph showing the activity of pre-cleaved (squares) and intact (circles) unmasked C-terminal half-life extended bivalent anti-STEAP-1 scFv / anti-CD3 Fab TCE (WW52322 / WW50870) in the LNCaP LDH cytotoxicity assay. The intact molecule shows no killing. The cleaved molecule begins to show some killing activity ; reaching max killing of 80% at tire concentrations tested. FIG. 25C is a graph showing the activity of an intact (squares) and pre-cleaved (circles) C-terminal half-life extended unmasked bivalent anti-STEAP-1 scFv / anti-CD3 scFv TCE (WW52453 / WW52454) in the LnCAP LDH cytotoxicity assay. The intact molecule shows no killing activity while the cleaved molecule begins to show killing activity at the higher concentrations tested.

[0161]

[0173] FIGs. 26A-26C are graphs showing the activity of T-cell engager prodrugs targeting CD3 and STEAP-1 as percentage of cytotoxicity. FIG. 26A is a graph showing the activity of intact (squares) and pre-cleaved (circles) C-terminal half-life extended ummasked anti-CD3 Fab / anti-STEAP- 1 scFv TCE (WW51875 / WW50870, anti-STEAP-1 scFv in VL / VH orientation) in the HEK293 LDH cytotoxicity assay. Neither intact or cleaved TCE shows killing activity. FIG. 26B is a graph showing the activity of pre-cleaved (circles) and intact (squares) unmasked C-terminal half-life extended bivalent anti-STEAP-1 scFv / anti-CD3 Fab TCE (WW52322 / WW50870) in the HEK293 LDH cytotoxicity assay. The intact molecule shows limited killing at the higher concentrations tested, while the cleaved molecule begins to show some killing activity with a predicted EC50 of 13pM. In FIG. 26C, the activity is shown for precleaved (circles) and intact (squares) unmasked C-terminal half-life extended bivalent anti-STEAP-1 scFv (VL / VH) / anti-CD3 scFv (VL / VH). TCE (WW52323 / WW52046) in the HEK293 LDH cytotoxicity assay. Both intact and pre-cleaved TCE demonstrate killing activity with EC50’s of 5pM. There is no steric blockade by the anti-HSA domain with this design.

[0162]

[0174] FIGs. 27A-27E are graphs showing the activity of T-cell engager prodrugs targeting CD3 and CDH-6 as percentage of cytotoxicity. FIG. 27A is a graph showing the activity of intact (circles) and precleaved (squares) C-terminal half-life extended unmasked anti-CD3 Fab / anti-CDH-6 scFv TCE (WW52009 / WW50870, anti-CDH-6 scFv in VH / VL orientation) in the OVCAR-3 HiBit cytotoxicity assay. There is a 4x window between intact and cleaved TCE, with the cleaved TCE having an EC50 of 6pM. FIG. 27B is a graph showing the activity of intact (circles) and pre-cleaved (squares) C-terminal half-life extended unmasked anti-CD3 / anti-CDH-6 DVD-Fab TCE (WW52017 / WW52018, long / long) in the OVCAR-3 HiBiT cytotoxicity assay. There is a 5x window between intact and cleaved TCE, with the cleaved TCE having an EC50 of 130pM. FIG. 27C is a graph showing the activity of intact (circles) and pre-cleaved (squares) C-terminal half-life extended unmasked anti-CDH-6 scFv (VH / VL) / anti-CD3 Fab TCE (WW52007 / WW50870) in the OVCAR-3 HiBit cytotoxicity assay. There is a 5x window between intact and cleaved TCE, with the cleaved TCE having an EC50 of 4.6pM. In FIG. 27D, a masked anti- CD3 scFv (VL / VH orientation) / anti-CDH-6 Fab with N-temiinal half-life extension and anti-CDH-6 masking using CDH-6 extracellular domain 3 (WW52097 / WW52099) demonstrates no target cell killing at high concentrations of TCE, circles. A pre-cleaved version of a similar TCE with a C-terminal half-life extension domain (squares) illustrates that removal of the anti-CD3 mask, half-life extension domain and anti-CDH-6 blocking domain leads to a potent TCE in the SKOV3 HiBiT cytotoxicity assay with an EC50 of 9pM. FIG. 27E is a graph showing a masked anti-CD3 scFv (VL / VH orientation) / anti-CDH-6 Fab TCE with C-terminal half-life extension and anti-CDH-6 masking using CDH-6 extracellular domain 3 (WW52097 / WW52099) that demonstrates no target cell killing at high concentrations of TCE, circles. A pre-cleaved version of tire same TCE (squares) illustrates that removal of the anti-CD3 mask, half-life extension domain and anti-CDH-6 blocking domain leads to a potent TCE in the SKOV3 HiBiT cytotoxicity assay with an EC50 of 55pM

[0163]

[0175] FIG. 28 is a graph showing binding of three anti-STEAP-1 T-cell engagers to HEK293 cells overexpressing human STEAP-1. A monovalent anti-CD3 Fab-anti-STEAP-1 scFv (VL / VH) with C tenninal half-life extension (WW51875 / WW50870, stars) show no binding to the HEK 293 cells. Hie full length vandortuzumab antibody (diamonds) shows excellent binding to the EIEK293 cells with an EC50 of 89pM. Both bivalent anti-STEAP scFv TCE’s (WW52322 / WW50870, squares and WW52323 / WW52046, circles) show equivalent binding to the HEK293 cells with EC50 values of 81pM and 134pm, respectively.

[0164]

[0176] FIGs. 29A-29C are schematic illustrations depicting half-life extended T-cell engager prodrugs that were tested in Example 24. FIG. 29A is a T-cell engager DVD Fab (long / long) prodrug comprising an antigen binding site for CD3 and an antigen binding site for EGFR, a half-life extension element, and a protease cleavable linker (WW51934 / WW51935). FIG. 29B is a T-cell engager DVD Fab (long / short) prodrug comprising an antigen binding site for CD3and an antigen binding site for EpCAM, a half-life extension element, and a protease cleavable linker (WW51938 / WW51937). FIG. 29C is a T-cell engager DVD Fab (short / long) prodrug comprising an antigen binding site for CD3 and an antigen binding site for B7H3. a half-life extension element, and a protease cleavable linker (WW51986 / WW51987).

[0165]

[0177] FIGs. 30A-30C are graphs showing the efficacy of half-life extended DVD Fab T-cell engager depicted in FIG. 29A using HCT-116 CDX model and total PBMC transfer as source for T effector cells. FIG. 30A is a graph showing average tumor volume vs time, comparing vehicle treated animals (circle) against half-life extended T-cell engager having an antigen binding site for EGFR (WW51934 / WW51935) (squares). FIGs. 30B and 30C are graphs showing tumor volume from individual animals from each treatment group, vehicle (FIG. 30B, circles) and WW51934 / WW 1935 (FIG. 30C, squares). Data demonstrate efficacy forWW51934 / WW51935 resulting in complete responses in most animals.

[0166]

[0178] FIGS. 31A-31D are graphs showing the efficacy of half-life extended DVD Fab T-cell engager depicted in FIG 29C using HCT-116 CDX model and total PBMC transfer as source for T effector cells. FIG. 31A is a graph showing average tumor volume vs time, comparing vehicle treated animals (circle) against half-life extended T-cell engager having an antigen binding site for B7-H3 (WW51936 / WW51955) administered at 3 pg (diamonds) and 10 pg (triangles). FIGs. 31B-31D are graphs showing tumor volume from individual animals from each treatment group, vehicle (FIG. 3 IB, circles), WW51936 / WW51955 at 3 ng (FIG. 31C, squares), and WW51936 / WW51955 at 10 qg (FIG. 31D, triangles). Data demonstrate efficacy for WW51936 / WW51955 at both 3 qg and 10 qg. Complete responses in most animals was observed at 10 pg.

[0167]

[0179] FIGS. 32A-32D are graphs showing the efficacy of half-life extended DVD Fab T-cell engagers depicted in FIG29B using HCT-116 CDX model and total PBMC transfer as source for T effector cells. FIG. 32A is a graph showing average tumor volume vs time, comparing vehicle treated animals (circle) against half-life extended T-cell engager having an antigen binding site for EpCAM (WW51938 / WW51937) administered at 3 qg (triangles) and 10 qg (diamonds). FIGs. 32B-32D are graphs showing tumor volume from individual animals from each treatment group, vehicle (FIG. 32B, circles), WW51938 / WW51937at 3 qg (FIG. 32C, triangles), and WW51938 / WW51937 at 10 qg (FIG. 32D, diamonds). Data demonstrate efficacy for WW51938 / WW51937 at both 3 qg and 10 qg with complete responses in most animals observed.

[0168]

[0180] FIGs. 33A-33C are graphs showing percent change in weight of animals over time for half-life extended DVD Fab T-cell engagers in FIGs. 30A-30C, FIGs. 31A-31C, and FIGs. 32A-32C. No weight loss greater than vehicle observed with treatment of half-life extended T-cell engager prodrugs, which indicate no signs of overt toxicity.

[0169]

[0181] FIGs. 34A-34C are schematic illustrations depicting half-life extended T-cell engager prodrugs that were tested in Example 25. FIG. 34A is a T-cell engager prodrug comprising an scFv having an antigen binding site for EGFR, a Fab comprising an antigen binding site for CD3, a half-life extension element, and a protease cleavable linker (WW51762 / WW50870). FIG. 34B is a T-cell engager prodrug comprising an scFv having an antigen binding site for EGFR, a Fab comprising an antigen binding site for CD3, a half-life extension element, and a protease cleavable linker (WW51758 / WW50870). FIG. 34C is a DVD Fab (long / long) T-cell engager prodrug comprising an antigen binding site for CD3and an antigen binding site for EGFR, a half-life extension element, and a protease cleavable linker (WW51934 / WW51935).

[0170]

[0182] FIGs. 35A-35E are graphs showing efficacy of half-life extended T-cell engagers depicted in FIGs. 34A-34C using HCT-116 CDX model and donor A as source for T effector cells. FIG. 35A is a graph showing average tumor volume overtime time, comparing vehicle treated animals against half-life extended T-cell engager prodrugs at lOug having binding specificity for EGFR. FIGs. 35B-35E are graphs showing tumor volume from individual animals from each treatment group, vehicle (FIG. 35B, circles), WW51758 / WW50870 (FIG. 35C, squares), WW51762 / WW50870 (FIG. 35D, triangles), and WW51934 / WW51935 (FIG. 35E, diamonds). Data demonstrate efficacy for each half-life extended T-cell engager prodrugs with complete responses observed for the scFv / Fab and DVD Fab (long / long) format.

[0171]

[0183] FIGs. 36A-36D are graphs showing percent change in weight of animals over time for half-life extended T-cell engagers in FIGs. 35A-35C. No weight loss greater than vehicle observed with treatment of half-life extended T-cell engager prodrugs, which indicate no signs of overt toxicity.

[0172]

[0184] FIGs. 37A-37E are graphs showing efficacy of half-life extended T-cell engagers depicted in FIGs. 34A-34C using HCT-116 CDX model and donor B as source for T effector cells. FIG. 37A is a graph showing average tumor volume overtime time, comparing vehicle treated animals against half-life extended T-cell engager prodrugs having binding specificity for EGFR. FIGs. 37B-37E are graphs showing tumor volume from individual animals from each treatment group, vehicle (FIG. 37B, circles), WW51758 / WW50870 (FIG. 37C, squares), WW51762 / WW50870 (FIG. 35D, triangles), and

[0173] WW5 1934 / WW51935 (FIG. 37E. diamonds). Data demonstrate efficacy for each half-life extended T-cell engager prodrug with complete responses observed for the scFv / Fab and DVD Fab (long / long) formats.FIGs. 38A-38D are graphs showing percent change in weight of animals overtime for half-life extended T-cell engagers in FIGs. 35A-35C. No weight loss greater than vehicle observed with treatment of half-life extended T-cell engager prodrugs, which indicate no signs of overt toxicity.

[0174] 4. DETAILED DESCRIPTION

[0175] A. Modified Antibodies or Antigen Binding Fragments

[0176]

[0185] The disclosure relates to modified antibodies or antibody binding fragments thereof (collectively referred to as modified ABs herein) that have inhibited binding activity for their cognate antigens. The modified ABs comprise at least a first antigen binding site (e.g., a VH / VL), and at least one and preferably two or more blocking elements that are operably linked through a cleavable linker (e.g., containing a disease-specific protease cleavage site) to the antigen binding site. The blocking elements, by being operably linked to the antigen binding site, can inhibit binding to the cognate antigen for example, by binding to the antigen binding site or sterically blocking the antigen binding site. The inventors have discovered, as described and exemplified herein, that operably linking two blocking elements to an antigen binding site surprisingly provides better inhibition of binding to the cognate antigen than a single blocking element. For example, such modified Abs in can be characterized as (a) have no or very little (e.g., less than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or substantially no) binding affinity or avidity for its cognate antigen or target (e.g., expressed on a cell) and / or (b) having no or very little (e.g., less than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or substantially no) functional activity (e.g. agonist, antagonist or inhibitory activity) on its cognate antigen or target (e.g., expressed on a cell. Upon cleavage of the protease cleavable linkers (e.g., protease cleavable linker peptide) the antigen binding domain can bind to its respective target. If desired, the modified ABs can further comprise an optional half-life extension element. When present, the half-life extension element can also function as a blocking element by sterically blocking the antigen binding site. Schematics of exemplary modified antibodies are illustrated in FIGs. 1A-1Z and 1ZA-1ZK.

[0177]

[0186] It will be appreciated by those skilled in the art, that the number of polypeptide chains, and the location of the elements, the modified ABs, the protease cleavable linker(s), tire blocking domains, and half-life extension element, when present, on the polypeptide chains can vary and is often a matter of design preference. All such variations are encompassed by this disclosure.

[0178]

[0187] The blocking elements, preferably two for each antigen binding site, can be operably linked to the modified ABs at any desired position to inhibit it from binding to its respective target and / or inhibit functional activity. The blocking elements can each independently be operably linked to the light chain and / or tire heavy chain. For example, the blocking elements, together with the protease cleavable linkers and optional half-life extension element can be operable linked to the N-temiinus of the antigen binding domain (e g., the N-terminus of the VH and or VL). In such examples, a single polypeptide that comprises, for example, from the N-terminal one or more blocking elements, a linker, optionally a halflife extension element and a protease cleavable linker and the VH or VL of the modified AB. This orientation is preferred when the blocking element is a peptide that comprises an epitope for tire modified AB that is located at the N-terminus of the cognate antigen or target of the modified AB. Other orientations, and modified ABs in which blocking elements (and optional half- life extension elements) are operably linked to the C-terminus of an antibody or antigen binding fragment (e.g., to the C-terminus of a VH. VHCH1. VL or VLCL) are also within the scope of this disclosure. The blocking elements can be operably linked to the amino (N)-terminus or carboxyl-(C) terminus of the heavy chain or light chain of the AB binding domain, or to combinations thereof. Preferably, each of the blocking elements are operably linked to the N-terminus of the heavy chain and / or light chain of the antigen binding domain.

[0179]

[0188] For example, the blocking elements can each independently be operably linked to the light chain of the antigen binding domain. The blocking elements can each independently be operably linked to the heavy chain of the antigen binding domain. A first blocking element can be operably linked to the heavy chain of the antigen binding domain and a second blocking element can be operably linked to the light chain of the antigen binding domain, and the first and second blocking elements can be the same or different.

[0180]

[0189] Tire blocking element can be operably linked to the antigen binding portion of the antibody light chain or tire antigen binding portion of the antibody of the heavy chain.

[0181]

[0190] The blocking element can be operably linked to the half-life extension element, when present.

[0182]

[0191] The blocking domains can sterically inhibit or block binding of the modified AB to its cognate antigen and / or functional activity induced by binding of the AB to its target. The blocking element that is a steric blocker can be a long half-life protein or peptide or a domain that binds to long half-life protein, such as serum albumin or transferrin. Suitable blocking elements of this type include, for example, a ligand binding domain or fragment of a cognate receptor, an antibody, or antibody fragment that binds to the desired target, such as serum albumin or transferrin. When the blocking element is an antibody or antigen-binding fragment, it can be, for example, a single domain antibody, a Fab. a scFv or other desired antigen binding fragment of an antibody. If desired at least one of the blocking elements can be human serum albumin, an antigen binding protein, or an antigen binding polypeptide which binds human serum albumin or a fragment thereof. The blocking element can optionally also extend the in vivo half-life when at least one of the blocking elements is human serum albumin or an antigen binding fragment thereof. Hie modified ABs can comprise more than two blocking elements (e.g., three blocking elements, four blocking elements, five blocking elements, or six blocking elements). For example, the modified ABs can comprise two blocking elements, three blocking elements, four blocking elements, five blocking elements, six blocking elements or more blocking elements.

[0183]

[0192] A preferred blocking element is a peptide that contains an epitope for a binding domain. Such peptides can comprise a conformational or linear epitope that is specifically bound by a binding domain. Peptides that comprise linear epitopes typically comprise at least about 8 amino acids, and frequently between about 8 amino acids and about 20 amino acids but can be longer or shorter if desired. For example the peptide can comprise about 5 to about 17 amino acids, for example about 5 to about 15 amino acids, about 7 to about 1 amino acids, about 5 to about 13 amino acids, about 7 to about 13 amino acids, about 8 to about 17 amino acids or about 8 to about 13 amino acids. Peptides blockers can comprise the amino acid sequence of tire immunogen used to create or isolate a binding domain. Peptide blockers can comprise an amino acid sequence that is the same as a region of the cognate antigen of the binding domain. Peptide blockers can also be any peptide that has binding affinity and specificity for the binding domain, such as engineered and synthetic peptides. In one example, a peptide that comprises the epitope for a binding domain is used to prepare a library of variant peptides. A peptide that binds to the binding domain with desired affinity can be identified and isolated from the library using suitable methods. In another example, peptides of random sequence can be designed or prepared and screed (e.g., in silico) to identify peptides that bind to a particular binding domain.

[0184]

[0193] A preferred blocking element is a peptide that contains an epitope for CD3epsilon. A preferred blocking element is a peptide that contains an epitope having SEQ ID NO: 496. A preferred blocking element is a peptide that contains an epitope having SEQ ID NO: 497. Such peptides can comprise a conformational or linear epitope that is specifically bound by a binding domain. Peptides that comprise linear epitopes typically comprise at least about 8 amino acids, and frequently between about 8 amino acids and about 20 amino acids but can be longer if desired.

[0185]

[0194] The blocking element can comprise a peptide that comprises or consists of the amino acid sequence of SEQ ID NO: 489. or an amino acid sequence that has at least 90% identity to SEQ ID NO:

[0186] 489.

[0187]

[0195] The blocking element can comprise a peptide that comprises or consists of the amino acid sequence of SEQ ID NO: 490, or an amino acid sequence that has at least 90% identity to SEQ ID NO:

[0188] 490.

[0189]

[0196] The blocking element can comprise a peptide that comprises or consists of the amino acid sequence of SEQ ID NO: 491. or an amino acid sequence that has at least 90% identity to SEQ ID NO:

[0190] 491.

[0191]

[0197] The blocking element can comprise a peptide that comprises or consists of the amino acid sequence of SEQ ID NO: 492, or an amino acid sequence that has at least 90% identity to SEQ ID NO:

[0192] 492.

[0193]

[0198] The blocking element can comprise a sequence that comprises or consists of the amino acid sequence of SEQ ID NO: 493. or an amino acid sequence that has at least 90% identity to SEQ ID NO:

[0194] 493.

[0195]

[0199] The modified Abs can comprise two or more blocking elements that also function as a half-life extension element. When two or more blocking elements are present in the modified Abs, a separate halflife extension element is optional and is typically not present.

[0196]

[0200] The modified Abs can optionally comprise one or more half-life extension elements. The half-life extension element can be, for example, a serum albumin binding domain, an antigen binding polypeptide which binds human serum albumin, a serum albumin, human serum albumin, transferrin, and an immunoglobulin Fc or fragment thereof, and the like. The half-life extension element can also contribute to inhibiting the binding of the modified AB to its cognate antigen, for example through steric effects. When the half-life extension element is absent, the blocking element can also function as a half-life extension element, if desirable (e.g., an antigen binding fragment of an antibody that binds HSA and sterically inhibits binding of the antigen binding domain to its respective target).

[0197]

[0201] When present, the one or more half-life extension elements can be operably linked to the one or more of the blocking elements, the first and / or second heavy chain antibody arm, the first and / or light chain antibody arm, the antigen binding portion of the heavy chain or the antigen binding portion of the light chain. The equilibrium dissociation constant (KD) of the blocking elements that specifically binds the antigen binding domain of the modified AB can be greater than the KDfor tire antigen binding domain towards the target. For instance, the KDof the blocking element for the antigen binding domain of the modified AB ca be at least 5, 10, 15, 20. 25. 50, 100, 250, 1000. 1500. 2000, 2500, 5000, 10000 or even greater than the KD for the antigen binding domain towards the target.

[0198]

[0202] The blocking elements that specifically binds the antigen binding domain of the modified AB generally bind to the antigen binding domain of the modified ABs with a KD of about 0.1 nM to about 50 pM.

[0199]

[0203] Preferably, the blocking elements that specifically binds the antigen binding domain of the modified AB generally bind to the antigen binding domain of the modified ABs with a KD of about 0.5 nM to about 10 nM. In some embodiments, the blocking elements bind the antigen binding domain of the modified ABs with a KD (KD = K .nfkdJ / K,,,, (ka)) of about 0.1 to 1 nM, about 0.1 to 5 nM, or about 10 to 50 nM, as determined by Bio-Layer Interferometry (BLI) or surface plasmon resonance (e.g., using a BIACORE instrument).

[0200]

[0204] In an example, tire modified AB can comprise a first blocking element and optionally a second blocking element operably linked through a protease cleavable linker to the heavy chain of the antigen binding domain. The blocking elements can be the same or different.

[0201]

[0205] In an example, the modified AB can comprise a first blocking element and optionally a second blocking element operably linked through a protease cleavable linker to the light chain of the antigen binding domain. Hie blocking elements and protease cleavable linkers can be the same or different.

[0202]

[0206] In an example, tire modified AB can comprise a first blocking element operably linked through a protease cleavable linker to the light chain of the antigen binding domain and a second blocking element operably linked through a protease cleavable linker to the heavy chain of the antigen binding domain. The blocking elements and protease cleavable linkers can be the same or different.

[0203]

[0207] In an example, the modified AB can comprise a first half-life extension element operably linked through a linker that is optionally protease cleavable to a first blocking element that is operably linked through a protease cleavable linker to the heavy chain of tire antigen binding domain, and a second halflife extension element operably linked through a linker that is optionally protease cleavable to a second blocking element that is operably linked through a protease cleavable linker to the light chain of the antigen binding domain. The first and second half-life extensions elements, the first and second blocking elements and protease cleavable and optionally protease cleavable linkers can be the same or different.

[0204]

[0208] In an example, the modified AB can comprise a first half-life extension element operably linked through a linker that is optionally protease cleavable to a first blocking element that is operably linked through a protease cleavable linker to the light chain of the antigen binding domain, and optionally include a second blocking element and / or second half-life extension element. The first and second halflife extensions elements, the first and second blocking elements and protease cleavable and optionally protease cleavable linkers can be the same or different.

[0205]

[0209] In an example, tire modified AB can comprise a first half-life extension element operably linked through a linker that is optionally protease cleavable to a first blocking element that is operably linked through a protease cleavable linker to the heavy chain of tire antigen binding domain, and optionally a second half-life extension element and / or second blocking element. The first and second half-life extensions elements, the first and second blocking elements and protease cleavable and optionally protease cleavable linkers can be the same or different.

[0206]

[0210] The modified ABs can comprise (i) a first binding domain (BD1), (ii) a first protease cleavable linker, (iii) a first inhibitory moiety (AIM) (e.g., a blocking element), which has a first activity’ inhibitory peptide (AIP1) that has affinity for the first binding domain (BD1) and a second activity inhibitory peptide (AIP2) that has affinity for the first binding domain (BD 1). The combination of the first activity inhibitory peptide (AIP1) and the second activity inhibitory peptide (AIP2) can provide a synergistic inhibitory activity to the first binding domain (BD1). The combination of the first activity inhibitory peptide (AIP1) and the second activity inhibitory peptide (AIP2) can provide inhibitory activity to the first binding domain (BD 1 ) that is at greater than the inhibitory activity of AIP 1 or AIP2 alone, for example, at least 10%. at least 20%. at least 30%, at least 40%, at least 50%, at least 60%. at least 70%. at least 80%, at least 90% or at least 95% greater than the inhibitory activity of AIP1 or AIP2 alone. The modified AB can further comprise an optional half-life extension element (XE).

[0207] [2H] Tire modified ABs can comprise (i) a first target antigen binding domain (BD1), (ii) a second target antigen binding domain (BD2), (iii) a first activity inhibitory moiety (AIM) that comprises a polypeptide sequence having a first activity inhibitory peptide (AIP1) having affinity for BD1 and a second activity inhibitory peptide (AIP2) having affinity for BD 1. The AIM can comprise an avidity (av) for BD 1 (AIM-avBD 1 ) greater than the sum of affinities for BD 1 of the first and second activity inhibitory peptides (AIP-avBDl +AIP2-avBDl), and (iv) a protease cleavable linker. The modified AB can further comprise an optional half-life extension element (XE).

[0208]

[0212] Tire modified ABs can comprise (i) a first target antigen binding domain (BD1). (ii) a second target antigen binding domain (BD2), (iii) a first activity inhibitory moiety (AIM) that comprises a polypeptide sequence having a first activity inhibitory peptide (AIP1) having affinity for BD1 and a second activity inhibitory peptide (AIP2) having affinity for BD1. The AIM can comprise an inhibitory activity (inh) for BD1 (AIM-inhBDl) greater than the sum of inhibitory activity for BD1 of the first and second activity' inhibitory' peptides (AIP-inhbitoryBD 1 +AIP2-inhibitoryBDl), and (iv) a protease cleavable linker. Hie modified AB can further comprise an optional half-life extension element (XE).

[0209]

[0213] The modified ABs can comprise (i) a first binding domain (BD1), (ii) a first protease cleavable linker, (iii) a first blocking element comprising a first activity inhibitory peptide (AIP 1 ) that has affinity for the first binding domain (BD1) and a second blocking element comprising second activity inhibitory peptide (AIP2) that has affinity for the first binding domain (BD 1). The combination of the first activity inhibitory peptide (AIP1) and the second activity inhibitory peptide (AIP2) can provide a synergistic inhibitory activity to the first binding domain (BD1). The combination of the first activity inhibitory peptide (AIP1) and the second activity inhibitory peptide (AIP2) can provide inhibitory activity to the first binding domain (BD1) that is at greater than the inhibitory activity of AIP 1 or AIP2 alone, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% greater than the inhibitory activity of AIP1 or AIP2 alone. Tire modified AB can further comprise an optional half-life extension element (XE).

[0210]

[0214] The modified ABs can comprise (i) a first target antigen binding domain (BD1). (ii) optionally a second target antigen binding domain (BD2), (iii) a first blocking element comprising a polypeptide sequence having a first activity inhibitory peptide (A1P1) having affinity for BD1 and a second blocking element comprising a polypeptide sequence having a second activity inhibitory peptide (AIP2) having affinity for BD 1 . The two blocking elements, together, can comprise an avidity (av) for BD 1 (blocking elements-avBDl) greater than the sum of affinities for BD1 of the first and second activity inhibitory peptides (AIP-avBDl +AIP2-avBDl), and (iv) a protease cleavable linker. The modified AB can further comprise an optional half-life extension element (XE).

[0211]

[0215] The modified ABs can comprise (i) a first target antigen binding domain (BD1). (ii) a second target antigen binding domain (BD2), (iii) a first blocking element comprising a polypeptide sequence having a first activity inhibitory peptide (AIP1) having affinity for BD1 and a second blocking element comprising a polypeptide sequence having a second activity inhibitory peptide (AIP2) having affinity for BD 1. The two blocking elements, together, can comprise an inhibitory activity (inh) for BD 1 (blocking elements-inhBDl) greater than the sum of inhibitory activity for BD1 of the first and second activity inhibitory peptides ( AIP-inhbitoryBD 1 +AIP2-inhibitoryBDl), and (iv) a protease cleavable linker. The modified AB can further comprise an optional half-life extension element (XE).

[0212]

[0216] The modified ABs of this disclosure can be a full-length antibody (e.g., IgG) or any desired antibody binding fragment containing an antigen binding domain, which is capable of binding to a target of interest. Examples of antibody binding fragments include, but are not limited to, variable regions of light and / or heavy chains of an antibody (VL, VH), variable fragments (Fv), Fab' fragments, F(ab') 2 fragments. Fab fragments, single chain antibodies (scAb), single chain variable regions (scFv), complementarity determining regions (CDR), domain antibodies (dAbs), single heavy chain immunoglobulins and variable domains thereof such as camelid VHH (e.g., nanobodies)and shark VNAR, single domain light chain immunoglobulins, and other polypeptides known in the art.

[0213]

[0217] Other various antibody and antigen-binding fragment fonnats are well known in the art and are encompassed by this disclosure. Exemplary antibody fonnats include, but are not limited to naked fullantibodies, naked antigen-binding fragments, Fc fusion proteins, multispecific antibodies (i.e., bispecific, trispecific, or tetraspecific), tetravalent antibodies: tetravalent monospecific antibodies, antibodyconjugates, radioimmunoglobulins, nanoparticles, diabodies, triabodies, minibody, monovalent, camelid or shark antibody.

[0214]

[0218] Antibody binding fragments retain the biological activity of an antibody (i.e., has the ability to bind and agonize its respective target). Biological assays can be used to confinn the antibody binding fragment has retained the biological function. Binding affinity is considered to be retained if it is approximately within about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold or about 10-fold the affinity of the corresponding antibody.

[0219] The modified ABs disclosed herein comprise at least a first antigen binding domain. The at least first antigen binding domain has binding specificity for any desired target antigen.

[0215]

[0220] Tire modified AB can comprise a first antigen binding domain that has specificity for any target antigen of interest. The first antigen binding domain can specifically bind to an antigen on a T-cell. Examples of suitable antigens on T-cells include, but are not limited to, CD3 epsilon, CD3 gamma, CD3 delta, CD19, CD20, CD45, CD28. CS2. CD3. CD4. LFA1, CD40. CD52, CD19, CD22. CD80, CD86, CD267, CD269, CD268, and CS32B. The first antigen binding domain can have specificity for the T-cell receptor complex. The first antigen binding domain with binding specificity for the TCR complex can specifically bind to CD3, preferably human CD3. Tire first antigen binding region can have binding specificity for one or more chain of the CD3 complex, such as CD3 epsilon, CD3 gamma, CD3 delta, or CD3 zeta. Hie first antigen binding region can have binding specificity for CD3 epsilon. The first antigen binding region can have binding specificity for CD3 gamma. The first binding region can have binding specificity for CD3 delta. The first binding region can have binding specificity for CD3 zeta.

[0216]

[0221] Suitable binding fragments that have specificity' for CD3 can be obtained, for example, from any anti-CD3 antibodies. Many such antibodies and binding regions are well-known, such as muromonab- CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35- 3, VIT3. BMA030 (BW264 / 56). CLB-T3 / 3. CRIS7. YTH12.5M, Fl 11-409, CLB-T3.4.2M TR-66, WT32, SPv-T3b, 11D8. XIII- 141. XIII-141. XIII-87, T3 / RW2-8C8. T3 / RW2-8C8, T3 / RW2-4B6. OKT3D, M- T301, SMC2, F101.01, UCHT-1, and WT-31.

[0217]

[0222] Additional antibodies having specificity for CD3 that can be suitable for the modified Abs include, but are not limited to, blinatumomab, solitomab, pasotuxizumab, mosunetuzumab, cevostamab, epcoritamab, tepoditamab, teclistamab, odronextamab, glofitamab, plamotamab, tebentafusp, duvortuxizumab, flotetuzumab, vixtimotamab, APVO436. HPN536. GBR1302, ERY974, GEMPSCA, A319. ONO-4685, elranatamab, SAR440234. TNB-383B, SP34, and muromonab.

[0218]

[0223] The first antigen binding domain that has specificity7for an antigen on a T-cell (e g., CD3) can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 999, a CDR2 having the amino acid sequence of SEQ ID NO: 1000, and a CDR3 having the amino acid of SEQ ID NO: 1001, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1002, a CDR2 having the amino acid sequence of SEQ ID NO: 1003, and a CDR3 having the amino acid of SEQ ID NO: 1004. Tire first antigen binding domain specificity for an antigen on a T-cell (e g., CD3) can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 995 and a light chain variable region having the amino acid sequence of SEQ ID NO: 996.

[0219]

[0224] Tire first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1005. a CDR2 having the amino acid sequence of SEQ ID NO: 1006, and a CDR3 having tire amino acid of SEQ ID NO: 1007, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1008, a CDR2 having the amino acid sequence of SEQ ID NO: 1009, and a CDR3 having the amino acid of SEQ ID NO: 1010. The first antigen binding domain specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 997 and a light chain variable region having the amino acid sequence of SEQ ID NO: 998.

[0220]

[0225] When present, the half-life extension element is preferably a sdAB that has binding specificity for human serum albumin and comprises a CDR1, a CDR2, and a CDR3. The CDR1 can comprise SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 37, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions. CDR2 can comprise SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 38, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions. CDR3 can comprise SEQ ID NO: 3. SEQ ID NO: 6. or SEQ ID NO: 12.

[0221]

[0226] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 863 and a light chain variable region comprising SEQ ID NO: 890.

[0222]

[0227] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 864 and a light chain variable region comprising SEQ ID NO: 891.

[0223]

[0228] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 865 and a light chain variable region comprising SEQ ID NO: 892.

[0224]

[0229] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 866 and a light chain variable region comprising SEQ ID NO: 893.

[0230] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 867 and a light chain variable region comprising SEQ ID NO: 894.

[0225]

[0231] Tire first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 868 and a light chain variable region comprising SEQ ID NO: 895.

[0226]

[0232] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 689 and a light chain variable region comprising SEQ ID NO: 896.

[0227]

[0233] Tire first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 870 and a light chain variable region comprising SEQ ID NO: 897.

[0228]

[0234] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 871 and a light chain variable region comprising SEQ ID NO: 898.

[0229]

[0235] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 872 and a light chain variable region comprising SEQ ID NO: 899.

[0230]

[0236] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 873 and a light chain variable region comprising SEQ ID NO: 900.

[0231]

[0237] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 874 and a light chain variable region comprising SEQ ID NO: 901.

[0232]

[0238] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 875 and a light chain variable region comprising SEQ ID NO: 902

[0233]

[0239] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 876 and a light chain variable region comprising SEQ ID NO: 903.

[0240] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 877 and a light chain variable region comprising SEQ ID NO: 904.

[0234]

[0241] Tire first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 878 and a light chain variable region comprising SEQ ID NO: 905.

[0235]

[0242] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 879 and a light chain variable region comprising SEQ ID NO: 906.

[0236]

[0243] Tire first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 880 and a light chain variable region comprising SEQ ID NO: 907.

[0237]

[0244] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 881 and a light chain variable region comprising SEQ ID NO: 908.

[0238]

[0245] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 882 and a light chain variable region comprising SEQ ID NO: 909.

[0239]

[0246] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 883 and a light chain variable region comprising SEQ ID NO: 910.

[0240]

[0247] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 884 and a light chain variable region comprising SEQ ID NO: 911.

[0241]

[0248] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 885 and a light chain variable region comprising SEQ ID NO: 912.

[0242]

[0249] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 886 and a light chain variable region comprising SEQ ID NO: 913.

[0250] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 887 and a light chain variable region comprising SEQ ID NO: 914.

[0243]

[0251] Tire first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 888 and a light chain variable region comprising SEQ ID NO: 915.

[0244]

[0252] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 889 and a light chain variable region comprising SEQ ID NO: 916.

[0245]

[0253] Other exemplary target antigens are described in Table 1.

[0246]

[0254] The protease cleavable linker can comprise the amino acid sequence selected from SEQ ID NOs: 510 to 535, 707. or 708.

[0247]

[0255] The one or more blocking elements having specificity for the first antigen binding domain (e.g., CD3) can be a peptide that contains an epitope for CD3 epsilon and can comprise the amino acid sequence of SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 496, and SEQ ID NO: 497, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 489. SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492. SEQ ID NO: 493, SEQ ID NO: 496, and SEQ ID NO: 497.

[0248]

[0256] The modified AB can comprise 1) a first polypeptide chain that comprises tire amino acid sequence of SEQ ID NO: 469; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0249]

[0257] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 461: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 470.

[0250]

[0258] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 472; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0251]

[0259] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 461: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 471.

[0260] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 469; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 470.

[0252]

[0261] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 469: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 471.

[0253]

[0262] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 472; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 470.

[0254]

[0263] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 460: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0255]

[0264] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 461; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 465.

[0256]

[0265] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 462: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0257]

[0266] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 461 and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 466.

[0258]

[0267] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 461: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 467.

[0259]

[0268] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 463; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0260]

[0269] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 461: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 468.

[0270] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 460; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 465.

[0261]

[0271] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 462: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 466.

[0262]

[0272] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 460; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 467.

[0263]

[0273] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 462: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 468.

[0264]

[0274] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 463; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 466.

[0265]

[0275] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 461: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0266]

[0276] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 482; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0267]

[0277] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 483: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 465.

[0268]

[0278] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 484; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0269]

[0279] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 483: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 466.

[0280] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 485; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0270]

[0281] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 483: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 467.

[0271]

[0282] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 485; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0272]

[0283] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 483: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 468.

[0273]

[0284] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 482; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 465.

[0274]

[0285] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 484: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 466.

[0275]

[0286] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 482; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 467.

[0276]

[0287] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 485: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 465.

[0277]

[0288] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 484; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 468.

[0278]

[0289] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 486: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 478.

[0290] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 483; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464.

[0279]

[0291] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 474: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 466.

[0280]

[0292] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 473; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 477.

[0281]

[0293] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 481 ; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 476.

[0282]

[0294] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 480; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 478.

[0283]

[0295] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 475: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 477.

[0284]

[0296] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 474; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 478.

[0285]

[0297] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 481 : and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 477.

[0286]

[0298] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 480; and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 476.

[0287]

[0299] The modified AB can comprise 1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 686: and 2) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 704.

[0300] Amino acid sequence variants of the modified ABs that have inhibited binding activity to their cognate antigens in the periphery and that release the antigen binding side upon cleavage of the disease specific protease at the desired target site (e.g., the tumor microenvironment or site of inflammation) can also be used in accordance with this disclosure. For example, the modified AB can include 1) a first polypeptide that comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%. at least about 98%, or at least about 99% identical to any one of SEQ ID Nos: 460-463, 469, 472, 473, 474, and 480- 486 and 2) a second polypeptide that comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs.: 464, 470, 471, 466, 467, 478, 476, and 477.

[0288] B. Immunoglobulin Formats

[0289]

[0301] The modified ABs disclosed herein can be formatted into a variety of suitable immunoglobulin formats. Immunoglobulin formats are well-known in the art and examples of suitable immunoglobulin fonnats are disclosed herein. For example, tire modified ABs provided herein can be monospecific or multispecific.

[0290]

[0302] The immunoglobulin formats disclosed herein can comprises a modified ABas disclosed herein, and optionally a linker (e.g., cleavable or non-cleavable linker). Suitable immunoglobulin formats are well-known in the art and examples of suitable immunoglobulin formats are disclosed herein. The immunoglobulin formats disclosed herein can contain at least two polypeptide chains, and can contain three or more polypeptide chains, if desired. Tire two or more polypeptide chains disclosed herein are different, i.e., the complexes can be heterodimers, heterotrimers, and tire like. Typically, immunoglobulin formats for bispecific T-cell engagers comprise two different polypeptide chains.

[0291]

[0303] The immunoglobulin formats disclosed herein can also be referred to as prodrug polypeptide(s) and / or multi-specific antibodies.

[0292]

[0304] In certain examples, the immunoglobulin format includes a bispecific, trispecific or multispecific cell engager, such as a BiTE, DART, BiKE or TriKE. Such prodrug polypeptides, e.g., T cell engager prodrugs, NK cell engager prodrugs, are described herein. In embodiments, the immunoglobulin format is structurally similar to such T cell engager or NK cell engager prodrugs, but omits the blocking elements. For example, the immunoglobulin format can comprise a sdAb or non-immunoglobulin format that binds HSA as described herein, and a binding region (sdAbs, scFv, Fab etc.) with binding specificity for an antigen on a T cell (e.g., CD3) and a binding region (sdAbs, scFv, Fab etc.) with binding specificity for an antigen on a target cell, such as a tumor associate antigen. In other examples, the immunoglobulin fragment can comprise a sdAb that binds HSA as described herein, and a binding region (sdAbs, scFv, etc.) with binding specificity for an antigen on aNK cell (e.g.. CD16) and a binding region (sdAbs, scFv, etc.) with binding specificity for an antigen on a target cell, such as a tumor associate antigen. As is well- known in the art, such bispecific, trispecific or multispecific cell engagers can comprise more than one polypeptide chain.

[0293]

[0305] The immunoglobulin formats disclosed herein can be linked directly or indirectly to the immune cell (e g., T cell orNK cell) binding domain and / or the target antigen (e.g., tumor associated antigen) binding domain. The immunoglobulin formats disclosed herein can be linked to the immune cell (e.g., T cell orNK cell) binding domain and / or the target antigen (e.g., tumor associated antigen) binding domain through a linker, such as a protease cleavable linker as described herein. The immunoglobulin formats disclosed herein can be linked to the immune cell binding domain. The immunoglobulin formats disclosed herein can be linked to the target antigen binding domain. The immunoglobulin formats disclosed herein can be linked to both the immune cell binding domain and the target antigen binding domain. Hie bispecific, trispecific or multispecific cell engager can comprise a) a sdAb or nonimmunoglobulin fonnat as disclosed herein that binds HSA. b) a binding region (e.g.. sdAb. scFv etc.) that has specificity for the TCR complex including CD3, and c) a binding region that has specificity for a target antigen, such as a tumor associated antigen.

[0294]

[0306] The binding region with specificity for the TCR complex can specifically bind to CD3, preferably human CD3. The binding region can have binding specificity for one or more chain of the CD3 complex, such as CD3 epsilon, CD3 gamma, CD3 delta, or CD3 zeta. The binding region can have binding specificity for CD3 epsilon. The binding region can have binding specificity for CD3 gamma. The binding region can have binding specificity for CD3 delta. The binding region can have binding specificity for CD3 zeta. The binding region having binding specificity for the TCR can further comprise a domain that specifically bind the alpha chain of the TCR and / or the beta chain of the TCR.

[0295]

[0307] The binding region having binding specificity to CD3 can be any suitable immunoglobulin format that binds CD3. Examples include, but are not limited to, an antigen binding fragments, such as single domain antibodies, Fab. Fab’. F(ab)2. single chain Fv fragments, disulfide stabilizd Fv fragments. Fv fragments, heavy chain monomers or dimers, light chain monomers or dimers. Suitable binding fragments that have specificity for CD3 can be obtained, for example, from any anti-CD3 antibodies. Many such antibodies and binding regions are well-known, such as muromonab-CD3 (0KT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5M, Flll-409, CLB-T3.4.2M TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII- 141, XIII-87, T3 / RW2-8C8, T3 / RW2-8C8, T3 / RW2-4B6, 0KT3D, M-T301, SMC2, F101.01. UCHT-1. and WT-31.

[0296]

[0308] In addition, the immunoglobulin format (e.g., bispecific, trispecific or multispecific cell engager) further comprises a region (e.g., sdAb, scFv, Fab) that binds to a target antigen. A target antigen is involved in and / or associated with a disease, disorder, or condition. In particular, the target antigen can be associated with or involved with a disease, disorder, or condition. Specifically, the target antigen can be associated with cancer, an inflammatory disease, an immunological disorder, an autoimmune disease, a viral disease, or a bacterial disease. The target antigen binding region can be any suitable immunoglobulin format that binds to the target antigen. Examples include, but are not limited to, a antigen binding fragments, such as single domain antibodies. Fab, Fab’, F(ab)2, single chain Fv fragments, disulfide stabilizd Fv fragments, pv fragments, heavy chain monomers or dimers, light chain monomers or dimers.

[0297]

[0309] Multispecific ABs provided herein bind more than one antigen. For instance, a multispecific antibody can bind two antigens (bispecific), three antigens (trispecific), four antigens, five antigens, or more. Many multispecific antibody formats and constructs are well-known in the art, and the modified ABs provided herein may be provided in the form of any suitable multispecific suitable construct. The multispecific antibody formats may comprise an antibody comprising at least two different heavy chain variable regions each paired with a common light chain variable region (i.e., a “common light chain antibody”). The common light chain variable region forms a distinct antigen-binding domain with each of the two different heavy chain variable regions. See Merchant et al., (1998), Nature Biotechnol., 16:677- 681. In some embodiments, the multispecific antibody formats comprises a modified AB, as described hereimattached to one or more of the N- or C-termini of the heavy or light chains of such immunoglobulin. See Coloma and Morrison, (1997), Nature Biotechnol., 15: 159-163. In some embodiments, the multispecific antibody formats comprises a hybrid immunoglobulin comprising at least two different heavy chain variable regions and at least two different light chain variable regions. See Milstein and Cuello (1983), Nature, 305:537-540; and Staerz and Bevan, (1986), Proc. Natl. Acad. Set. USA, 83: 1453-1457.

[0310] The multispecific antibody formats provided herein can comprise immunoglobulin chains with alterations to reduce the formation of side products that do not have multi specificity. Preferably, the multispecific antibody formats provided herein comprise one or more “knobs-into-holes” modifications, as described in U.S. Pat. No. 5,731,168. The multivalent antibody formats provided herein can comprise immunoglobulin chains with one or more electrostatic modifications to promote the assembly of Fc hetero-multimers. See WO 2009 / 089004. In other embodiments, the multivalent antibody fomrat can comprise a bispecific single chain molecule. See Traunecker et al., (1991), EMBO J., 10:3655-3659; and Gruber et al., (1994), J. Immunol., 152:5368-5374.

[0298] [3H] In some embodiments, the multivalent antibody formats provided herein comprises a heavy chain variable domain and a light chain variable domain connected by a polypeptide linker, where the length of the linker is selected to promote assembly of multispecific antibody fomrats with the desired multispecificity. For example, monospecific scFvs generally form when a heavy chain variable domain and light chain variable domain are connected by a polypeptide linker of more than 12 amino acid residues. See U.S. Pat. Nos. 4,946,778 and 5,132,405. In some embodiments, reduction of the polypeptide linker length to less than 12 amino acid residues prevent pairing of heavy and light chain variable domains on the same polypeptide chain, thereby allowing pairing of heavy and light chain variable domains from one chain with the complementary domains on another chain. The resulting antibody formats therefore have multispecificity, with the specificity of each binding site contributed by more than one polypeptide chain. Polypeptide chains comprising heavy and light chain variable domains that are joined by linkers between 3 and 12 amino acid residues form predominantly dimers (termed diabodies). With linkers between 0 and 2 amino acid residues, trimers (termed triabodies) and tetramers (termed tetrabodies) are favored. However, the exact type of oligomerization appears to depend on the amino acid residue composition and the order of the variable domain in each polypeptide chain (e.g., VH-linker- VLvs. VL-linker-VH). in addition to the linker length. A skilled person can select the appropriate linker length based on the desired multispecificity.

[0299]

[0312] In some embodiments, the multispecific antibody formats provided herein can comprise a diabody. See Hollinger et al., (21993), Proc. Natl. Acad. Sci. USA, 90:6444-6448. The multivalent antibody formats can also comprise a triabody or a tetrabody (Todorovska et al., (2001). J. Immunol. Methods, l^Al-66), s a tetrabody(Id ), or a trispecific F(ab')3 derivative (Tutt et al., (1991) J. Immunol.. 147:60-69.

[0313] In some embodiments, the multispecific antibody formats provided herein comprises a crosslinked antibody. See U.S. Pat. No. 4,676,980; Brennan et al., (1985), Science, 229:81-83; Staerz, et al., (1985), Nature, 314:628-631; and EP 0453082.

[0300]

[0314] In some embodiments, the multispecific antibody fonnats provided herein comprises antigenbinding domains assembled by leucine zippers. See Kostelny et al., (1992), J. Immunol., 148: 1547-1553.

[0301]

[0315] As provided herein, the multivalent antibody formats may also comprise complementary protein domains. Preferably, the complementary protein domains comprise an anchoring domain (AD) and a dimerization and docking domain (DDD). Generally, the AD and DDD bind to each other and thereby enable assembly of multispecific antibody structures via tire “dock and lock” (DNL) approach.

[0302] Multispecific antibody formats of many specificities may be assembled, including bispecific multispecific antibodies, trispecific antibodies, tetraspecific antibodies, quintspecific antibodies, and hexaspecific antibodies. Multispecific antibodies comprising complementary protein domains are described in the art, for example, in U.S. Pat. Nos. 7,521,056; 7,550,143; 7,534,866; and 7,527,787.

[0303]

[0316] In some embodiments, the multispecific antibody formats comprise a dual action Fab (DAF) antibody as described in U.S. Pat. Pub. No. 2008 / 0069820. The multivalent antibody formats can comprise an antibody formed by reduction of two parental molecules followed by mixing of the two parental molecules and reoxidation to assembly a hybrid structure. See Carlring et al., (2011), PLoS One, 6:e22533.

[0304]

[0317] In a preferred embodiment, the multivalent antibody formats provided herein comprises a tetravalent bispecific antibody. The tetravalent bispecific antibody may be comprising an antibody, an antigen-binding fragment, or a combination of both. In some embodiments, the tetravelent bispecific antibody comprises an antibody and an antigen-binding fragment. Alternatively, the tetravalent bispecific antibody can also comprise two antibodies or two antigen-binding fragments.

[0305]

[0318] In some embodiments, the multispecific antibody formats comprise an antigen-binding fragment attached to another antibody or antigen-binding fragment. The attachment can be covalent or non- covalent. When the attachment is covalent, it may be in the form of a fusion protein or via a chemical linker. Illustrative examples of multispecific antibody formats comprising antibody fragments attached to other antibodies include tetravalent bispecific antibodies, where an scFv is fused to the C-terminus of the CH3 from an IgG. See Coloma and Morrison, (1997), Nature Biotechnol., 15: 159-163. Other examples include antibodies in which a Fab molecule is attached to the constant region of an immunoglobulin. See Miler et al., (2003), J. Immunol., 170:4854-4861. Any suitable fragment may be used, including any of the fragments described herein or known in the art.

[0306]

[0319] Tire multispecific antibody formats can be in a variety of formats that are well-known in the art. Exemplary multispecific antibody formats include, but are not limited to, a DVD-Ig™ (see U.S. Pat. No.

[0307] 7,612,181) a DART™ (e.g., Moore et al., (2011), Blood, 117:454-451), DuoBody®, (Labrijn et al., (2013), Proc. Natl. Acad. Set. USA, 110:5145-5150; Gramer etal., (2013), mAbs, 5:962-972; and Labrijn et al., (2014), Nature Protocols, , 9:2450-2463), a CovX-Body ( Doppalapudi et al., (2010), Proc. Natl. Acad. Set. USA, 107:22611-22616), an Fcab antibody(Wozniak-Knopp et al., (2010), Protein Eng. Des. Sei., 23:289-297), a TandAb® antibody (Kipriyanov et al., (1999), J. Mol. Biol., 293:41-56 and Zhukovsky et al., (2013), Blood, 122:5116), a tandem Fab, which are described in WO 2015 / 103072, or Zybody™ (LaFleur et al., (2013). mAbs, 5:208-218).

[0308]

[0320] This disclosure also relates to modified ABs as described herein in which that antigen binding domain of an antibody is replaced with a binder that includes a non-immunoglobulin scaffold. Any suitable alternative scaffold may be used. Exemplary alternative scaffold that are well-known include, but are not limited to, Adnectin™, iMab, Anticalin®, EETI-II / AGRP, Kunitz domain, thioredoxin peptide aptamer, Affibody®, DARPin, Affilin. Tetranectin, Fynomer, and Avimer, Aflfimers, Alphabodies, Atrimers, Avimers, Centyrins, Obodies, Pronectins, and Repebodies.

[0309]

[0321] The modified ABs disclosed herein can comprise two or more antigen binding domains. In some examples, the two or more antigen binding domains each independently bind to different target antigens. For example, the first and second antigen binding domains can each bind to a tumor antigen. In some examples, the tw o or more antigen binding domains bind to the same target.

[0310]

[0322] Examples of a first and second target antigen binding domains include, but are not limited to, a T cell engager, a bispecific T cell engager, a dual-affinity re-targeting antibody, a variable heavy domain (VH), a variable light domain (VL). a scFv comprising a VH and a VL domain, a soluble TCR fragment comprising a Valpha and Vbeta domain, a single domain antibody (sdAb), or a variable domain of camelid derived nanobody (VHH), a non-Ig binding domain.

[0311]

[0323] Provided herein is an isolated multi-specific antibody comprising: (a) a first polypeptide complex comprising at least a first and a second polypeptide sequence, wherein the first polypeptide sequence comprises P1-LP1 -BD1L (Formula I), and wherein the second polypeptide sequence comprises P1-LP1- BD1H-BD1CH (Formula 11). and (b) a second polypeptide complex comprising at least a third and a fourth polypeptide sequence, wherein the first polypeptide sequence comprises P2-LP2-BD2L (Formula Ill), and wherein the second polypeptide sequence comprises P2-LP2-BD2H-BD2CH (Formula IV). BD1L and BD1H form BD1, comprising a CD 3 binding domain. BD2L and BD2H form BD2, comprising a target antigen binding domain. LP1 and LP2 independently comprise a protease cleavable linker peptide. Pl comprises a first activity inhibitory peptide (AIP 1) having an affinity for BD 1. P2 comprises a second activity inhibitory peptide (AIP2) having an affinity for BD2. The first polypeptide sequence can comprise P1-XD-LP1-BD1L (Formula la). XD comprises a half-life extension domain. The second polypeptide sequence can comprises? I -XD-LP I -BD1H-BD1 CH (Formula Ila). XD comprises a half-life extension domain. The third polypeptide sequence can comprise P2-XD-LP2-BD2L (Formula Illa). XD comprises a half-life extension domain. Tire fourth polypeptide sequence comprises P2-XD- LP2-BD2H-BD2CH (Formula IVa). XD comprises a half-life extension domain.

[0312]

[0324] Provided herein is isolated multi-specific antibody comprising, in any order: BD 1 comprising a CD3 binding domain; BD2 comprising a target antigen binding domain; LI and L2 independently comprising a linker peptide; LP comprising a protease cleavable linker peptide; Pl comprising a first activity inhibitory peptide (AIP1) having an affinity for BD1; P2 comprising a second activity inhibitory peptide (AIP2) having an affinity for BD1; optionally, P3 comprising a third activity inhibitory peptide (AIP3) having an affinity for BD2; and optionally. P4 comprising a fourth activity inhibitory peptide (AIP4) having an affinity for BD2.

[0313] C. T-cell and NK cell engagers

[0314]

[0325] In certain examples, the immunoglobulin format includes bispecific, trispecific, or multispecific T-cell engagers, such as a BiTE, DART, BiKE or TriKE. The T-cell engager can comprise a modified AB as disclosed herein, and a target antigen binding domain. For example, the T-cell engager can comprise (i) a first antigen binding domain that has specificity for an antigen on a T-cell (e,g, CD3) (e.g.. sdAbs, scFv, Fab etc.), (ii) preferably two or more blocking elements that have binding specificity for the first antigen binding domain that are operably linked through a cleavable linker to the first antigen binding domain, (iii) an optional half-life extension element, and (iv) a target antigen binding domain (e.g., sdAbs, scFv, Fab etc.). If desired, the two or more blocking elements that have binding specificity for the first antigen binding domain can be omitted.

[0315]

[0326] The T-cell engager can further comprise an optional blocking element that has binding specificity to the target antigen binding domain. The T-cell engager can comprise one or more blocking elements that has binding specificity to the target antigen binding domain. If desired, the T-cell engager can comprise more than two blocking elements (e.g., three blocking elements, four blocking elements, five blocking elements, or six blocking elements). The blocking element, when present, can be operably linked to an antigen binding domain (e.g., the first antigen binding domain) or target antigen binding domain through a protease cleavable linker. The blocking element can inhibit binding of the target antigen to its target for example, by binding to the antigen binding domain or sterically blocking tire antigen binding domain.

[0316]

[0327] The T-cell engager can comprise a modified AB as disclosed herein that comprises two or more blocking elements having binding specificity to the first antigen binding domain (e.g., CD3) and at least one blocking element that has binding specificity to the target antigen binding domain.

[0317]

[0328] When the modified AB is formatted into a T-cell engager, the first antigen binding domain preferably has specificity for an antigen on a T-cell, such as the TCR or a component of the TCR or a CAR. Preferably, the first antigen binding domain can specifically bind to one or more chains of the CD3 complex, such as CD3 epsilon, CD3 gamma, CD3 delta, or CD3 zeta. Hie modified AB can also be formatted into an NK cell engager, and the first antigen binding domain preferably has specificity for an antigen on aNK cell, such as CD 16a or NKp46.

[0318]

[0329] The target antigen binding domain has specificity for any target of interest. Exemplary' target antigens are described in Table 1. Preferred target antigens include, EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, and STEAP-1. For example, a preferred target antigen is EGFR. For example, a preferred target antigen is B7-H3. For example, a preferred target antigen is DLL-3. For example, a preferred target antigen is CDH6. For example, a preferred taiget antigen is EpCAM. For example, a preferred target antigen is STEAP-1 .

[0319]

[0330] The target binding domain can be a full-length antibody (e.g., IgG) or any desired antibody binding fragment containing an antigen binding domain, which is capable of binding to a target of interest. Examples of antibody fragments include but are not limited to, variable regions of light and / or heavy chains of an antibody (VL, VH), variable fragments (Fv). Fab' fragments. F(ab') 2 fragments. Fab fragments, single chain antibodies (scAb), single chain variable regions (scFv), complementarity determining regions (CDR), domain antibodies (dAbs), single heavy chain immunoglobulins and variable domains thereof such as camelid VHH (e.g., nanobodies) and shark VNAR, single domain light chain immunoglobulins, and other poly peptides known in the art.

[0320]

[0331] In some instance, the target binding domain can be non-Ig binding domains antibody mimetic, such as Adnectin™. iMab. Anticalin®, EETI-1I / AGRP, Kunitz domain, thioredoxin peptide aptamer, Affibody®, DARPin, Affilin, Tetranectin, Fynomer, and Avimer, Affimers, Alphabodies, Atrimers, Avimers, Centyrins, Obodies, Pronectins, and Repebodies.

[0321]

[0332] In some instances, the target binding domain can be a cognate receptor or fragment thereof of the target antigen or a ligan binding domain or fragment thereof.

[0322]

[0333] The T-cell engagers can further comprise one or more additional target antigen binding domains. For example, two target antigen binding domains, three target antigen binding domains, four target antigen binding domains, or more.

[0323]

[0334] In certain preferred T-cell engagers, the first antigen binding domain is a Fab fragment, or a single chain variable region (scFv). In certain preferred T-cell engagers, the target antigen binding domain is a Fab fragment or a single chain variable region (scFv). Typically, only one of the first antigen binding domains or target binding antigens is a Fab fragment and only one of the first antigen binding domains or target binding domains us a scFv. For example, when the first antigen binding domain is a Fab fragment, the target antigen binding domain can be a scFv. For example, when the first antigen binding domain is a scFv, the target antigen binding domain can be a Fab.

[0324]

[0335] As is well-known in the art, such bispecific, trispecific or multispecific T-cell engagers can comprise more than one polypeptide chain.

[0325]

[0336] Tire T-cell engagers disclosed herein can contain at least two polypeptide chains, and can contain three or more polypeptide chains, if desired. The two or more polypeptide chains disclosed herein are different, i.e., the complexes can be heterodimers, heterotrimers, and the like. Typically, T-cell engager for bispecific T-cell engagers comprise two different polypeptide chains.

[0326]

[0337] Components that fomr the first antigen binding domain are typically present on separate polypeptide chains. For example, a first polypeptide chain can include an antibody light chain (VL+CL), or light chain variable domain (VL) and a second polypeptide can include an antibody heavy chain Fab fragment (VH + CHI) or heavy chain variable domain (VH) that is complementary to the VL+ CL or VL on the first polypeptide. In such situations, these components can associate in the peptide complex to form the first antigen binding domain.

[0327]

[0338] Components of the target antigen blocking element can be present on separate polypeptide chains. For example, a first polypeptide chain can include an antibody light chain (VL+ CL), or light chain variable domain (VL) and a second polypeptide can include an antibody heavy chain Fab fragment (VH + CHI) or heavy chain variable domain (VH) that is complementary to the VL+ CL or VL on the first polypeptide. In such situations, these components can associate in the peptide complex to form a binding site for the target antigen binding domain.

[0328]

[0339] For example a first polypeptide chain can include components of the first antigen binding domain (e.g., VL + CL or VL) and / or components of the target antigen binding domain (e.g., VL + CL or VL) (e.g., operably linked through a linker), and complementary components of the first antigen binding domain (e.g., VH + CHI or VH) and / or the target antigen binding domain (e.g., VH + CHI or VH) can be present on the one or more other polypeptide chains polypeptide chain (e.g. both on a separate second polypeptide chain and operably linked through a linker). For example, when the T cell engager comprises a Fab binding domain, one polypeptide chain (e.g, a “first” polypeptide chain) can comprise heavy chain components of the Fab and other components of the T cell engager as described herein, and a separate polypeptide (e.g, a “second” polypeptide chain) can comprise light chain components of the Fab and other components of tire T cell engager as described herein. The two chain can associate through covalent (e.g., disulfide and / or non-covalent bonds).

[0329]

[0340] The second polypeptide chain can further comprise a half-life extension element. The half-life extension element can be operably liked through a linker to the components of the first antigen binding domain (e g., linked to VH or CHI) or components of the target antigen binding domain (e.g., linked to VH or CHI). Preferably, the linker is a protease cleavable linker, as described herein. The half-life extension element can be operably linked to the N-terminus or C-terminus of the desired polypeptide, such as the second polypeptide chain. When the first antigen binding domain or the target antigen binding domain comprises a Fab, the half-life extension element can be operably linked to CHI.

[0330]

[0341] Tire first polypeptide chain can further comprise a half-life extension element. The half-life extension element can be operably liked through a linker to tire components of the first antigen binding domain (e g., VL + CL or VL) or components of the target antigen binding domain (e.g., VL + CL or VL). Preferably, tire linker is a protease cleavable linker, as described herein. The half-life extension element can be operably linked to the N-terminus or C-terminus of the first polypeptide chain. When the first antigen binding domain or the target antigen binding domain comprise a VL + CL, the half-life extension element can be operably linked to CL at the C-terminus. The half-life extension element can be operably linked through a linker to tire first blocking element and a component of the first antigen binding domain or target antigen binding domain.

[0342] Typically, only the first polypeptide chain or the second polypeptide chain will comprise a halflife extension element. Those of skill in the art will appreciate that, if desired, the first polypeptide chain and second polypeptide chain can each independently comprise a half-life extension element.

[0331]

[0343] Two or more blocking elements having binding specificity to the first antigen binding domain (e.g., CD3), when present, can be present on either the first polypeptide chain or second polypeptide chain, or both the first polypeptide chain and second polypeptide chain. For example, the two blocking elements can be present on the first polypeptide chain. For example, the two blocking elements can be present on the second polypeptide chain. For example, one blocking element can be on the first polypeptide chain, and one blocking element can be on the second polypeptide chain.

[0332]

[0344] A first blocking element having binding specificity to tire first antigen binding domain (e.g., CD3) can be present on the second polypeptide. The first blocking element can be operably linked to the components of the first antigen binding domain (e.g., VH + CHI or VH) or components of the target antigen binding domain (e.g., VH + CHI or VH) through a linker. Preferably, the first blocking element is operably linked to the components of the first antigen binding domain. Preferably, tire linker is a protease cleavable linker, as described herein. The first blocking element is preferably operably linked to the C- terminus of tire second polypeptide.

[0333]

[0345] A second blocking element having binding specificity to the first antigen binding domain can be present on the first polypeptide chain. Tire second binding element can be operably linked to the components of the first antigen binding domain (e.g., VL + CL or VL) or components of the target antigen biding domain (e.g., VL + CL or VL) through a linker. Preferably, the linker is a protease cleavable linker as described herein. Preferably, the second blocking element is operably linked to the components of the first antigen binding domain. The second blocking element is preferably operably linked to the C- terminus of the first polypeptide.

[0334]

[0346] The optional one or more blocking elements that have binding specificity to the target antigen binding domain can be present on the first polypeptide chain, the second polypeptide chain, or both the first polypeptide chain and second polypeptide chain.

[0335]

[0347] For example, a blocking element having binding specificity to the target antigen binding domain can be present on the second polypeptide chain. Tire blocking element can be operably linked to the components of the first antigen binding domain (e.g., VH + CHI or VH) or components of the target antigen binding domain (e.g., VH + CHI or VH). Preferably, the blocking element is operably linked to the components of the tumor antigen binding domain through a linker that is optionally a protease cleavable linker. For example, a blocking element having binding specificity to the target antigen binding domain can be present on the first polypeptide chain. The blocking element can be operably linked to the components of the first antigen binding domain (e.g., VL + CL or VL) or components of the target antigen binding domain (e.g., VL + CL or VL). Preferably, the blocking element is operably linked to the components of the tumor antigen binding domain through a linker that is optionally a protease cleavable linker. For example, a first blocking element can be operably linked to the half-life extension element, when present, through a linker that is optionally a protease cleavable linker.

[0336]

[0348] When the T-cell engager comprises two or more blocking elements having binding specificity to the target antigen binding domain, the two or more blocking elements can be present on the first polypeptide chain, the second polypeptide chain, or both the first polypeptide chain and second polypeptide chain (e.g., one blocker on the first chain and one blocker on the second chain).

[0337]

[0349] Schematics of exemplary T-cell engagers are illustrated in FIGs. 3A-3F. 17A-17P. and 18A-18H. Schematics of exemplary T-cell engagers are also illustrated in FIGs. 19A-19L 20A-20L 29A-29C, and 34A-34B.

[0338]

[0350] For example, as shown in FIGs. 3A and 3B, tire T-cell engager can comprise 1) a first polypeptide chain that comprises VH + CHI or VH, a blocking element having binding specificity for the first antigen binding domain (e.g., CD3 epsilon), a half-life extension element, a target antigen binding domain, a target antigen blocking element, and a protease cleavable linker: and 2) a second polypeptide chain comprising an chain comprising a VL+CL or VL that is complementary to the VH + CHI or VH. The VH + CHI or VH and VL+CL or VL associate in the peptide complex and form the first binding site.

[0339]

[0351] For example, as shown in FIG. 3C, the T-cell engager can comprise 1) a first polypeptide chain that comprises VH + CHI or VH, a blocking element having binding specificity for the first antigen binding domain (e.g., CD3 epsilon), a half-life extension element, a target antigen binding domain, and a protease cleavable linker, 2) a second polypeptide chain comprising a VL+CL or VL that is complementary to the VH + CHI or VH that is operably linked through linker to a target antigen blocking element. The VH + CHI or VH and VL+CL or VL associate in the peptide complex and form the first binding site.

[0340]

[0352] For example, as shown in FIGs. 3D and 3E, the T-cell engager can comprise 1) a first polypeptide chain that comprises VH + CHI or VH, a first blocking element having binding specificity for the first antigen binding domain (e.g., CD3 epsilon), a half-life extension element, a target antigen binding domain, a target antigen blocking element and a protease cleavable linker, 2) a second polypeptide chain comprising a VL+CL or VL that is complementary to the VH + CHI or VH that is operably linked through a protease cleavable linker to second blocking element having binding specificity for the first antigen binding domain (e.g., CD3 epsilon). The VH + CHI or VH and VL+CL or VL associate in the peptide complex and form the first binding site. Tire VH + CHI or VH and VL+CL or VL associate in the peptide complex and fonn the first binding site.

[0341]

[0353] For example, as shown in FIG. 3F, tire T-cell engager can comprise 1) a first polypeptide chain that comprises VH + CHI or VH, a first blocking element having binding specificity for the first antigen binding domain (e.g., CD3 epsilon), a half-life extension element, a target antigen binding domain, and a protease cleavable linker, 2) a second polypeptide chain comprising a VL+CL or VL that is complementary to the VH + CHI or VH, a second blocking element having binding specificity for the first antigen binding domain (e.g., CD3 epsilon), a target antigen blocking element, and a protease cleavable linker. The VH + CHI or VH and VL+CL or VL associate in the peptide complex and form the first binding site. The VH + CHI or VH and VL+CL or VL associate in the peptide complex and form the first binding site.

[0342]

[0354] For example, as shown in FIGs. 17A-17D, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH, (ii) a VH + CHI, (iii) a linker that operably links the VH and the VH + CHI, (iv) an optional half-life extension element, and (v) when the half-life extension element is present, a protease cleavable linker that operably links the half-life extension element and the VH + CHI, and 2) a second polypeptide chain that comprises (i) a VL that is complimentary to the VH, (ii) a VL + CL that is complementary to the second VH + CHI, and (iii) a linker that operably links the first VL and tire second VL + CL. Tire VH and the VL can associate in the peptide complex to form a first antigen binding domain (e.g., CD3 epsilon), and the VH + CHI and the VL + CL can associate in the peptide complex and fonn a target antigen binding domain (e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1). The VH and the VL can associate in the peptide complex to form a target antigen binding domain (e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1), and the VH + CHI and the VL + CL can associate in the peptide complex a first antigen binding domain (e.g., CD3 epsilon). In FIGs. 17A-17D, the two or more blocking elements, as described hereon, that are operably linked through a cleavable linker to the antigen binding domains are omitted. When the half-life extension element is present, it can be operably linked to the C-terminus of the VH + CHI . Various linkers can be used to operably link the VH to the VH + CH 1 and / or the VL to the VL + CL 1. Tire linker can be a short linker, a long linker, or combinations thereof, as described herein.

[0355] For example, as shown in FIGs. 17E-17H, the T-cell engager can comprise 1) a polypeptide chain that comprises (i) a VH, (ii) a first blocking element that is operably linked to the VH through a cleavable linker element having binding specificity for the first antigen binding domain (e.g., CD3 epsilon), (iii) a VH + CHI, (iv) a linker that operably links the VH and the VH + CHI, (v) optionally a half-life extension element, and (vi) when the half-life extension element is present, a protease cleavable linker that operably links tire half-life extension element and the VH + CHI. and 2) a second polypeptide chain that comprises (i) a VL that is complimentary to the VH, (ii) a second blocking element that is operably linked to the VL element having binding specificity for the antigen binding domain (e.g., CD3 epsilon), (iii) a VL + CL that is complementary to the VH + CHI, and (iv) a linker that operably links the VL and the VL + CL. Hie VH and the VL can associate in tire peptide complex to form a first antigen binding domain (e.g., CD3 epsilon), and the VH + CHI and the VL + CL can associate in the peptide complex and fonn a target antigen binding domain (e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1). The VH and the VL can associate in the peptide complex to form a target antigen binding domain (e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1), and the VH + CHI and the VL + CL can associate in the peptide complex and form a first antigen binding domain (e.g., CD3 epsilon). When the half-life extension element is present, it can be operably linked to the C-terminus of the VH + CHI .

[0343] Various linkers can be used to operably link the VH to the VH + CHI and / or the VL to the VL + CL1. Tire linker can be a short linker, a long linker, or combinations thereof, as described herein.

[0344]

[0356] For example, as shown in FIG. 171, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH + VL (e.g., a scFv) that associate and form a target antigen binding domain, (ii) a VH + CH, (iii) an optional half-life extension element, and (iv) when the half-life extension element is present, a protease cleavable linker that operably links the half-life extension element and the VH + CHI, and 2) a second polypeptide chain that comprises (i) a VL +CL that is complementary to the VH + CHI . The VH + CHI and the VL + CL associate in the peptide complex and form an antigen binding domain (e.g., CD3 epsilon). When the half-life extension element is present, it can be operably linked to the C- terminus of the VH + CHI .

[0345]

[0357] For example, as shown in FIG. 17J, the T-cell engager comprise 1) a first polypeptide chain that comprises (i) a VH + VL (e.g., a scFv) that fonn a target antigen binding domain, (ii) a first blocking element having binding specificity for a first antigen binding domain, (iii) a protease cleavable linker that operably links the first blocking element to the VH + VL that forms a target antigen binding domain, (iv) a VH + CHI, (v) an optional half-life extension element, and (vi) when the half-life extension element is present, a protease cleavable linker that operably links the half-life extension element and the VH + CHI, and 2) a second polypeptide chain that comprises (i) a VL + CL that is comprises (i) a VL + CL that is complementary to the VH + CHI, (ii) a second blocking element having binding specificity for a first antigen binding domain, and (ii) a protease cleavable linker that operably links the second blocking element to the VL + CL. Hie VH + CHI and the VL + CL associate in the peptide complex and fomi an antigen binding domain (e.g., CD3 epsilon). When the half-life extension element is present, it can be operably linked to the C-terminus of the VH + CHI .

[0346]

[0358] For example, as shown in FIG. 17K, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH + CHI, (ii) a VH + VL (e.g., a scFv) that form a target antigen binding domain operably linked to the CHI, (iii) optionally a half-life extension element, and (iv) when the half-life extension element is present, a protease cleavable linker that operably links the half-life extension element and the VH + VL (e.g., scFv). and 2) a second polypeptide chain that comprises (i) a VL + CL that is complementary to the VH + CHI . The VH + CHI and the VL + CL associate in the peptide complex and form an antigen binding domain (e.g., CD3 epsilon). When the half-life extension element is present, it can be operably linked to the C-terminus of the VH + CHI .

[0347]

[0359] For example, as shown in FIG. 17L. the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH + VL (e.g., a scFv) that form a target antigen binding domain, (ii) a VH + CHI, (iii) a first blocking element having binding specificity a first antigen binding domain, (iv) a protease cleavable linker that operably links the first blocking element to the VH +CH1, (v) optionally a half-life extension element, (vi) when the half-life extension element is present, a protease cleavable linker that operably links the half-life extension element and the VH + VL, and 2) a second polypeptide chain that comprises (i) a VL + CL that is complementary to the VH + CHI, (ii) a second blocking element having binding specificity to a first antigen binding domain, and (iii) a protease cleavable linker that operably links tire second blocking element to the VL + CL. The VH + CHI and the VL + CL associate in tire peptide complex and form a first antigen binding domain (e.g., CD3 epsilon). When the half-life extension element is present, it can be operably linked to the C-terminus of the VH + CHI.

[0348]

[0360] For example, as shown in FIG. 17M, the T-cell engager can comprise 1) a first polypeptide that comprises (i) a VH + VL (e.g., a scFv) that fonn a first antigen binding domain (e g., CD3 epsilon), (ii) a VH + CHI, (iii) optionally a half-life extension element, and (iv) when the half-life extension element is present, a protease cleavable linker that operably links the half-life extension element and the VH + CHI. and 2) a second polypeptide chain that comprises (i) a VL + CL that is complementary' to the VH + CHI . The VH + CHI and the VL + CL associate in the peptide complex and form a target antigen binding domain (e.g., e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1). When the half-life extension element is present, it can be operably linked to the C-terminus of the VH + CHI.

[0349]

[0361] For example, as shown in FIG. 17N, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH + VL (e.g. scFv) that fonns a first antigen binding domain (e.g., CD3 epsilon), and (ii) a VL + CL, and 2) a second polypeptide chain that comprises (i) a VH + CHI. (ii) an optional half-life extension element, and (iii) when the half-life extension element is present, a protease cleavable linker that operably links the half-life extension element and tire VH + CHI . The VH + CHI and the VL + CL associate in the peptide complex and form a target antigen binding domain (e.g., e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1).

[0350]

[0362] For example, as shown in FIG. 170, the T-cell engager can comprise 1) a first polypeptide chain that comprises i) a VH + VL (e.g.. a scFv) that fonns a first antigen binding domains (e.g., CD3 epsilon), ii) a VH + CH, ii) a first blocking element having specificity for the first antigen binding domains, iii) a protease cleavable linker that operably links the first blocking element with the VH + VL, iv) optionally a half-life extension element, v) when tire half-life extension element is present, a protease cleavable linker that operably links the half-life extension element to the VH + CHI, and 2) a second polypeptide chain that comprises a VL + CL. ii) a second blocking element having specificity for the first antigen binding domain, iii) a protease cleavable linker that operably links the second blocking element with the VL + CL. The VH + CHI and the VL + CL associate in the peptide complex and form a target antigen binding domain (e.g., e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1). When the half-life extension element is present, it can be operably linked to the C-terminus of the VH + CHI.

[0351]

[0363] For example, as shown in FIG. 17P, the T-cell engager can comprises 1) a first polypeptide chain that comprises (i) a VH + CHI, (ii) a first blocking element that has binding specificity for a first antigen binding domain (e.g., CD3 epsilon), (iii) a half-life extension element, and (iv) a protease cleavable linker that operably links the half-life extension element and the first blocking element the VH + CHI, and 2) a second polypeptide chain that comprises (i) a VL + CL that is complementary to the VH + CHI, (ii) VH + VL that forms a first antigen binding domain (e.g., CD3 epsilon), (iii) a second blocking element having specificity for the first antigen binding domain, and (iv) a protease cleavable linker that operably links tire second blocking element to the VH + VL.

[0352]

[0364] For example, as shown in FIGs. ISA, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH, (ii) a VH + CHI, (iii) a linker that operably links the VH and the VH + CHI, (iv) a half-life extension element, and (v) a protease cleavable linker that operably links the half-life extension element and the VH + CH 1 , (vi) a blocking element having specificity for a target antigen binding domain, and 2) a second polypeptide that comprises (i) a VL that is complimentary to the VH, (ii) a VL + CL that is complementary to the VH + CHI, and (iii) a linker that operably links the VL and the VL + CL. The VH and the VL can associate in the peptide complex to form a first antigen binding domain (e.g., CD3 epsilon), and the VH + CHI and the VL + CL can associate in the peptide complex and form a target antigen binding domain (e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1). The VH and the VL can associate in the peptide complex to form a target antigen binding domain (e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1), and the VH + CHI and the VL + CL can associate in the peptide complex a first antigen binding domain (e.g., CD3 epsilon). In FIG. 18A, the two or more blocking elements having specificity for the first antigen binding domain, described herein, are omitted. Various linkers can be used to operably link tire VH to the VH + CHI and / or the VL to the VL + CL1. The linker can be a short linker, a long linker, or combinations thereof, as described herein.

[0365] For example, as shown in FIGs. 18B and 18G, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH, (ii) a VH + CHI, (iii) a linker that operably links tire VH and the VH + CHI, (iv) a half-life extension element, (v) a first protease cleavable linker that operably links the half-life extension element and tire VH + CHI, (vi) a blocking element having specificity for a target antigen binding domain, (vii) a first blocking element having specificity for a first antigen binding domain, and (vii) a second protease cleavable linker that operably links the first blocking element to the first VH, and 2) a second polypeptide that comprises (i) a VL that is complimentary to the VH, (ii) a VL + CL that is complementary to the VH + CHI, (iii) a linker that operably links the VL and the VL + CL, (iv) a second blocking element having specificity for a first antigen binding domain, and (vii) a third protease cleavable linker that operably links the second blocking element to the VL. The VH and the VL can associate in the peptide complex to form the first antigen binding domain (e.g., CD3 epsilon), and the VH + CHI and the VL + CL can associate in the peptide complex and form a target antigen binding domain (e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1). If desired, the immunoglobulin fomrat can omit the second blocking element and / or tire third blocking element having binding specificity for the first antigen binding domain, as depicted in FIG. 18H. Various linkers can be used to operably link the VH to the VH + CHI and / or the VL to the VL + CL1. The linker can be a short linker, a long linker, or combinations thereof, as described herein.

[0366] For example, as shown in FIGs. 18C, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH, (ii) a VH + CHI, (iii) a linker that operably links the VH and the VH + CHI, (iv) a half-life extension element, (v) a first protease cleavable linker that operably links the half-life extension element and the VH + CHI, (vi) a blocking element having specificity for a target antigen binding domain, (vii) a second protease cleavable linker that operably links the blocking element to the VH, and 2) a second polypeptide chain that comprises (i) a VL that is complimentary to the VH, (ii) a VL + CL that is complementary to the VH + CHI, (iii) a linker that operably links the VL and the VL + CL. The VH and the VL can associate in the peptide complex to form a target antigen binding domain (e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1). The VH + CHI and the VL + CL can associate in the peptide complex and form an antigen binding domain (e g., having binding specificity for CD3 epsilon). The half-life extension element is operably linked to the C-terminus of the VH + CHI. Various linkers can be used to operably link the VH to the VH + CHI and / or the VL to the VL + CL1. Tire linker can be a short linker, a long linker, or combinations thereof, as described herein.

[0353]

[0367] For example, as shown in FIG. 18D, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH, (ii) a VH + CHI, (iii) a linker that operably links the VH and the VH + CHI, (iv) a half-life extension element, (iii) a first blocking element having specificity for a target antigen binding domain, (iv) a second blocking element having specificity for a first antigen binding domain, (v) a first protease cleavable linker that operably links the half-life extension element and the VH + CHI, and (vi) a second protease cleavable linker that operably links the first blocking element and the VH, and 2) a second polypeptide chain that comprises (i) a VL that is complimentary to the VH, (ii) a VL + CL that is complimentary to the VH + CHI, (iii) a third blocking element that has binding specificity for the target antigen binding domain, and (iv) a third protease cleavable linker that operably links the third blocking element to the VL. The VH and the VL can associate in the peptide complex to form a target antigen binding domain (e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1). The VH + CHI and the VL + CL can associate in the peptide complex and form a antigen binding domain (e.g., having binding specificity for CD3 epsilon). The half-life extension element is operably linked to the C-terminus of the VH + CHI . Various linkers can be used to operably link tire VH to the VH + CHI and / or the VL to the VL + CL1. Tire linker can be a short linker, a long linker, or combinations thereof, as described herein.

[0354]

[0368] For example, as shown in FIG. 18E. the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH + VL (e.g., a scFv) that form a target antigen binding domain, (ii), a VH + CHI, (iii), a half-life extension element, (iv), a first blocking element having specificity for a target antigen binding domain, (v) a second blocking element having specificity for a first antigen binding domain, (vi) a first protease cleavable linker that operably links the half-life extension element to the VH + VL, and (vii) a second protease cleavable linker that operably links the second blocking element to the VH + CH, and 2) a second polypeptide chain that comprises (i) a VL + CL that is complimentary to the VH + VL, (ii) a third blocking element that has binding specificity for the first antigen binding domain, and (iii), a protease cleavable linker that operably links the blocking element to the VL + CL. The VH + CHI and the VL + CL can associate in the peptide complex and form the antigen binding domain (e.g., having binding specificity for CD3 epsilon). If desired, the immunoglobulin format can omit the second blocking element and / or the third blocking element having binding specificity for the antigen binding domain, as depicted in FIG. 18F.

[0355]

[0369] For example, as shown in FIG. 18G, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH. (ii). a VH + CHI. (iii), a half-life extension element, (iv). a first blocking element having specificity for a target antigen binding domain, (v) a second blocking element having specificity for a first antigen binding domain, (vi) a first protease cleavable linker that operably links the half-life extension element to the VH + CHI, (vii) a second protease cleavable linker that operably links the second blocking element to the VH, and (viii) a linker that operably links the VH and the VH + CHI; and 2) a second polypeptide chain that comprises (i) a VL that is complimentary to the VH, (ii) a VL + CL that is complimentary to the VH + VL, (ii) a third blocking element that has binding specificity for the first antigen binding domain, and (iii), a protease cleavable linker that operably links the blocking element to the VL. The VH + VL can associate in the peptide complex and form the first antigen binding domain (e.g., e.g., having binding specificity for CD3 epsilon), and the VH + CHI and VL + CL can associate in the peptide complex and form a target antigen binding domain (e.g., EGFR, B7-H3, DLL-3, CDH6, EpCAM, PSMA, or STEAP-1). If desired, the immunoglobulin format can omit the second blocking element and / or the third blocking element having binding specificity for the antigen binding domain, as depicted in FIG. 18H.

[0356]

[0370] For example, as shown in FIG. 20A and 20B, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) two or more VH + VL (e.g., a scFv) that fonn two or more target antigen binding domains (e.g., EGFR, B7-H3, DLL-3. CDH6, PSMA, EpCAM, or STEAP-1), for example a first VH + VL that forms a first target antigen binding domain, a second VH + VL that forms a second target antigen binding domain, and if desired, a third VH + VL that forms a third target antigen binding domain, (ii), a VH + CHI, (iii), a half-life extension element (vi) a first protease cleavable linker that operably links the half-life extension element to the VH + CHI; (vii) a first blocking element having specificity for a first antigen binding domain, and (v) a first protease cleavable linker that operably links the first blocking element to tire VL + CL; and 2) a second polypeptide chain that comprises (i) a VL + CL that is complimentary to tire VH + CHI, (ii) a second blocking element having specificity for the first antigen binding domain, and (iii) a second protease cleavable linker that operably links tire second blocking element to the VH + CHI . The VH + CHI and the VL + CL can associate in the peptide complex and form the antigen binding domain (e.g., having binding specificity for CD3 epsilon). If desired, the immunoglobulin format can omit the first blocking element and / or the second blocking element having binding specificity for the antigen binding domain, as depicted in FIGs. 19A an 19B. The immunoglobulin format can comprise two VH +VL that form target antigen binding domains. The immunoglobulin fonnat can comprise three VH + VL that form target antigen binding domain. The target antigen binding domains can have binding specificity for the same target antigen. The target antigen binding domains can have binding specificity for different target antigens. The half-life extension element can be operably linked to the C-terminus of the VH + CHI .

[0357]

[0371] For example, as shown in FIG. 20C, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) two or more VH + VL (e.g., two or more scFv’s) that form two or more target antigen binding domains (e.g.. EGFR, B7-H3, DLL-3. CDH6. PSMA. EpCAM, or STEAP-1), for example a first VH + VL that forms a first target antigen binding domain, and a second VH + VL that forms a second target antigen binding domain, (ii), CHI, (iii), a half-life extension element (vi) a first protease cleavable linker that operably links the half-life extension element to the CHI, (vii), a first blocking element having binding specificity for a first antigen binding domain on the second polypeptide chain, and (viii) a protease cleavable linker that operably links the first blocking element to one of the VH + VL; and 2) a second polypeptide chain that comprises i) a VH + VL (e.g.. a scFv) that fonn an antigen binding domain (e.g., having binding specificity for CD3 epsilon), (ii) a CL (e.g., that is complimentary to the CHI), (iii) a second blocking element having specificity for the first antigen binding domain, and (iv) a protease cleavable linker that operably links the second blocking element to the VH + VL that forms an antigen binding domain. The half-life extension element can be operably linked to the C-terminus of the VH + CHI. If desired, the immunoglobulin fonnat can omit the first blocking element and / or the second blocking element having binding specificity for the antigen binding domain, as depicted in FIG. 19C.

[0358]

[0372] For example, as shown in FIG. 20D, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a VH + VL (e.g., a scFv) that forms a target antigen binding domains (e.g., EGFR, B7- H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1), (ii) a VH + CHI, (in) a half-life extension element, (vi) a first blocking element having binding specificity for a first antigen binding domain on the second polypeptide chain, (vii) a first protease cleavable linker that operably links the half-life extension element to the first blocking element; and 2) a second polypeptide chain that comprises (i) a VL + CL that is complimentary to the VH + CHI, (ii) a VH + VL (e.g., a scFv) that form a first antigen binding domain (e.g., having binding specificity for CD3 epsilon), (ii) a second blocking element having specificity for the first antigen binding domain, and (iv) a protease cleavable linker that operably links the second blocking element to the VH + VL that forms a first antigen binding domain. The VH + CHI and VL + CL can associate in the peptide complex and form a second target antigen binding domain (e.g., EGFR, B7- H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1). The first target antigen binding domain and the second target binding domain can have binding specificity for the same target antigen. The first target antigen binding domain and the second target antigen binding domain can have binding specificity for different target antigens.

[0359] If desired, the immunoglobulin format can omit the first blocking element and / or the second blocking element having binding specificity for the antigen binding domain, as depicted in FIG. 19D.

[0360]

[0373] For example, as shown in FIGs. 20E and 20F, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a first VH + VL (e.g., a scFv) that forms a first target antigen binding domains (e.g.. EGFR, B7-H3, DLL-3. PSMA. CDH6. EpCAM, or STEAP-1), (ii) a second VH + VLthat forms a second target antigen binding domain, (ii) a VH + CHI, (iii) a half-life extension element, (vi) a first blocking element having binding specificity for a first antigen binding domain, (vii) a first protease cleavable linker that operably links the half-life extension element to the first blocking element; and 2) a second polypeptide chain that comprises (i) a VL + CL that is complimentary to the VH + CHI, (ii) a second blocking element having specificity for the first antigen binding domain, and (iii) a protease cleavable linker that operably links the second blocking element to the VH + VL. The VH + CHI and VL + CL can associate in the peptide complex and form a first antigen binding domain (e.g., CD3). The first target antigen binding domain and target antigen binding domain can have binding specificity for the same target antigen. The first target antigen binding domain and target antigen binding domain can have binding specificity for different target antigens. The first VH + VL can be operably linked to the C- terminus of the VH + CHI . The second VH + VL can be operably linked to the VH of the VH + CHI or the first VH + VL. If desired, the immunoglobulin format can omit the first blocking element and / or the second blocking element having binding specificity for the antigen binding domain, as depicted in FIGs. 19E and 19F.

[0361]

[0374] For example, as shown in FIGs. 20G, 20H, and 201, the T-cell engager can comprise 1) a first polypeptide chain that comprises (i) a first VH + VL (e.g., a scFv) that forms a first target antigen binding domains (e.g., EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1), (ii) an optional second VH + VL that forms a second target antigen binding domain, (iii) a VH + CHI, (iv) a half-life extension element, (v) a first blocking element having binding specificity for a first antigen binding domain on the second polypeptide chain, (vi) a first protease cleavable linker that operably links the half-life extension element to tire first blocking element, and (vii) optionally a protease cleavable linker that operably links the half-life extension element to the VH of the VH + CHI or the first or second VH + VL, if present, and 2) a second polypeptide chain that comprises (i) a third VH + VL (e g., a scFv) that forms a third target antigen binding domain, (ii) a VL + CL that is complimentary to the VH + CHI, (iii) a second blocking element having specificity for the first antigen binding domain, and (iv) a protease cleavable linker that operably links the second blocking element to the VL + CL that forms an antigen binding domain. The VH + CHI and VL + CL can associate in the peptide complex and form the first antigen binding domain (e.g. CD3 epsilon). The first target antigen binding domain, the optional second target antigen binding domain, and third target antigen binding domain can have binding specificity for tire same target antigen or different target antigens. The first VH + VL can be operably linked to the C-tenninus of the VH + CHI . The third VH + VL can be operably linked to the C-terminus of the VL + CL. The optional second VH + VL, when present, can be operably linked to the first VH + VL or the VH of the VH + CHI . If desired, the immunoglobulin format can omit the first blocking element and / or the second blocking element having binding specificity for the antigen binding domain, as depicted in FIGs. 20G-20I. Various linkers can be used to operably link the VH to the VH + CHI and / or the VL to the VL + CL. Tire linker can be a short linker, a long linker, or combinations thereof, as described herein.

[0362]

[0375] The bispecific T-cell engagers can also comprise three different polypeptide chain, if desired.

[0363]

[0376] The T-cell engagers can further comprise a binding region with specificity to a co-stimulatory signaling molecule. Tire binding region can have binding specificity for any desired co-stimulatory molecule. Exemplary co-stimulatory molecules are well known and include, but are not limited to, CD28, 4-1BB, ICOS, CD27, CD2, CD40L. GITR, 0X40 and CD226. The binding region can have binding specificity for CD28. The binding region can have binding specificity for 4-1BB. The binding region can have binding specificity for ICOS. The binding region can have binding specificity for CD26.

[0377] The T-cell engagers can further comprise a binding region with specificity for an immune checkpoint inhibitor. The binding region can have binding specificity for any desired immune check-point inhibitor. Exemplary immune checkpoint inhibitors are well known and include, but are not limited to 2B4, TIGIT, CD155, ICOS, HVEM, LIGHT, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD40, CEACAM1. CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, ID02, TDO, KIR, LAG- 3, TIM-3, and VISTA.

[0364]

[0378] The modified Abs and immunoglobulin formats disclosed herein can be linked directly or indirectly to the immune cell (e.g., T cell or NK cell) binding domain and / or tire target antigen (e.g., tumor associated antigen) binding domain. The modified ABs disclosed herein can be linked to the immune cell (e.g., T cell or NK cell) binding domain and / or the target antigen (e.g., tumor associated antigen) binding domain through a linker, such as a protease cleavable linker as described herein. The modified ABs disclosed herein can be linked to the immune cell binding domain. The modified ABs disclosed herein can be linked to the target antigen binding domain. The modified ABs disclosed herein can be linked to both the immune cell binding domain and the target antigen binding domain.

[0365] D. Targets of Modified Antibody Binding Constructs

[0366]

[0379] As disclosed herein, the modified Abs and immunoglobulin formats can further comprise a second binding domain that has binding specificity for a second target antigen of interest. The second target antigen of interest can be associated with, for example, but not limited to, a cancer, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disorder, a viral disease, or a parasitic disease.

[0367]

[0380] Exemplary target antigens include those listed in Table 1, which is not exhaustive. The target antigen can be a tumor antigen expressed on a tumor cell. Tumor antigens are well known in the art and include, for example, EpCAM, EGFR, CTLA4, PD-1, PDL-1 , 4-1BB, HER-2, HER-3, c-Met, FoIR, PSMA, CD38, BCMA, CEA, 5T4, AFP, B7-H3, CDH-6, CAIX, CD117, CD123, CD1238, CD166, CD18, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3. GCP3, gpA33, FLT-3, gpNMB, HPV-16, E6. HPV-16 E7, ITGA2, ITGA3. SLC39A6, MAGE, mesothelin, Mucl, Mucl6. NaPi2b, Nectin-4, CDH-3, CDH-17, EPHB2, ITGAV, ITGB6, NY-ESO-1, PRLR, PSCA, PTK7, R0R1, SLC4A4, SLTRK5, SLITRK6, STEAP1, TIMI, Trop2, claudin (e.g., claudin 1, claudin 4, claudin 6, claudin 7, claudin 18.2), or WT1. Certain preferred tumor antigens include, but are not limited to, EGFR, B7-H3, DLL3, CDH-6, EpCAM, and STEAP-1.

[0368]

[0381] Tire target antigen can be an immune checkpoint protein. Exemplary immune checkpoint protein includes, but is not limited to CD27, CD137, 2B4, TIGIT, CD155. ICOS, HVEM, CD40L, LIGHT, 0X40, DNAM-1, PD-L1. PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDOL IDO2, TDO, KIR, LAG-3, TIM-3, and VISTA.

[0369]

[0382] The target antigen can be a cell surface molecule such as a protein, lipid or polysaccharide. The second target antigen can be on a tumor cell, virally infected cell, bacterially infected cell, damaged red blood cell, arterial plaque cell, inflamed or fibrotic tissue cell.

[0370]

[0383] Tire target antigen can be CD19, CD22, CD30, CD33. CD79b, HER2, trop2, nectin4, tissue factor of folate receptor alpha.

[0371]

[0384] The target antigen can be a tumor antigen selected from EGFR, PSMA, EpCAM, BCMA. 5T4, KLK2, AFP, Axl, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD38, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, CEACAM5, c-MET, DLL3, EphA2, FAP, FGFR2, FGFR3, glypican-3, FLT-3, FOLR1, gpNMB, HER2, HPV-16 E6, HPV-16 E7, ITGA3, SLC39A6, Mesothelin, Mucl. Mucl6, NaPi2b, Nectin-4, P-cadherin, Prolactin R, PSCA, PTK7, RORL SLC44A4, SLTRK5, SLTRK6. STEAPL TIM1, Trop2, and WT1. Tire target antigen can be a tumor antigen or immunomodulatory protein tumor selected from CTLA-4, CD27. CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, 0X40, DNAM-1, PD-L1, PD1, PD- L2, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, VISTA, IL6-R, IL-6, TNFa, CD 19, CD20, CD22, CD52, integrin a4, integrin a4b7, CDlla, CTLA4-Ig fusion, IL-17, IL12 / 23, IL12, IL23, TGF-beta, IL-2, IL-21, IL-18, IL2RA, IL2RB, IL2RG, IL21R, IL18R1, IL18RAP, IL12RBL IL12RB2, CD4, 4-1BB, 4-1BBL. GITR, GITRL and a claudin. such as claudin 1. claudin 4, claudin 6, claudin 7 or claudin 18.2.

[0372]

[0385] Preferably the target antigen is a tumor antigen. Preferred tumor antigens suitable for the modified Abs and immunoglobulin formats include EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, and STEAP-1.

[0373] Table 1. Exemplary targets antigens

[0374]

[0375]

[0386] The target antigen binding domain having specificity for EGFR and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 959, a CDR2 having the amino acid sequence of SEQ ID NO: 960, and a CDR3 having the amino acid of SEQ ID NO: 961, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 962, a CDR2 having the amino acid sequence of SEQ ID NO: 963, and a CDR3 having the amino acid of SEQ ID NO: 964. The target antigen binding domain specificity for EGFR can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 947 and a light chain variable region having the amino acid sequence of SEQ ID NO: 948.

[0376]

[0387] The target antigen binding domain having specificity for B7-H3 and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 965, a CDR2 having the amino acid sequence of SEQ ID NO: 966, and a CDR3 having the amino acid of SEQ ID NO: 967, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 968, a CDR2 having the amino acid sequence of SEQ ID NO: 969, and a CDR3 having the amino acid of SEQ ID NO: 970. The target antigen binding domain having specificity B7-H3 (e.g., CD3) can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 949 and a light chain variable region having the amino acid sequence of SEQ ID NO: 950.

[0377]

[0388] The target antigen binding domain having specificity for CDH6 and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 977, a CDR2 having the amino acid sequence of SEQ ID NO: 978, and a CDR3 having the amino acid of SEQ ID NO: 979, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 980, a CDR2 having the amino acid sequence of SEQ ID NO: 981, and a CDR3 having the amino acid of SEQ ID NO: 982. The target antigen binding domain having specificity for CDH6 can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 953 and a light chain variable region having the amino acid sequence of SEQ ID NO: 954.

[0378]

[0389] The target antigen binding domain having specificity for EpCAM and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 983, a CDR2 having the amino acid sequence of SEQ ID NO: 984, and a CDR3 having the amino acid of SEQ ID NO: 985, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 986, a CDR2 having the amino acid sequence of SEQ ID NO: 987, and a CDR3 having the amino acid of SEQ ID NO: 988. The target antigen binding domain having specificity for EpCAM can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 955 and a light chain variable region having the amino acid sequence of SEQ ID NO: 956.

[0379]

[0390] The target antigen binding domain having specificity for STEAP-1 and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 989, a CDR2 having the amino acid sequence of SEQ ID NO: 990, and a CDR3 having the amino acid of SEQ ID NO: 991, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 992, a CDR2 having the amino acid sequence of SEQ ID NO: 993, and a CDR3 having the amino acid of SEQ ID NO: 994. The target antigen binding domain having specificity for STEP-1 can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 957 and a light chain variable region having the amino acid sequence of SEQ ID NO: 958.

[0380]

[0391] The target antigen binding domain having specificity for DLL and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 971, a CDR2 having the amino acid sequence of SEQ ID NO: 972, and a CDR3 having the amino acid of SEQ ID NO: 973, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 974. a CDR2 having the amino acid sequence of SEQ ID NO: 975, and a CDR3 having the amino acid of SEQ ID NO: 976. The target antigen binding domain having specificity for DLL can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 951 and a light chain variable region having the amino acid sequence of SEQ ID NO: 952.

[0381] E. Blocking Element

[0382]

[0392] In the present disclosure, the modified Abs or immunoglobulin formats comprises two or more blocking elements that specifically or sterically blocks binding of the modified AB to its target. The blocking element can be operably linked to the modified ABs by one or more protease cleavable linkers, as described herein. Upon cleavage of tire protease linker the antigen binding domain(s) disassociate from the modified AB and thereby allowing the modified ABs to bind its targets. One exemplary blocking element is serum albumin (e.g., human serum albumin (HSA)), or anti-HSA antibodies. Blocking elements may be attached to antibody light chains, heavy chains, or both. Two or more blocking elements can be used (e.g., 2, 3, 4, 5, or more). The number of blocking elements will depend on the number of exposed antigen binding arms. The one or more blocking domains can be the same or different.

[0383]

[0393] Tire blocking element can be any element that inhibits the ability of the antigen binding domains to bind and / or activate its target. Examples of suitable blocking elements include a chain of the cognate receptor of the target antigen.

[0384]

[0394] Antibodies and antigen-binding fragments thereof that bind the antigen binding domains, including an antigen-binding fragment (Fab), a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid- type nanobody (VHH), a dAb and the like, can also be used as blocking elements. Other suitable antigenbinding domains can also be used, including non-immunoglobulin proteins that mimic antibody binding and / or structure such as. Adnectin™, iMab, Anticalin®. EETI-II / AGRP. Kunitz domain, thioredoxin peptide aptamer, Affibody ®, DARPin, Affdin, Tetranectin, Fynomer, and Avimer, Affimers, Alphabodies, Atrimers, Avimers, Centyrins, Obodies, Pronectins, and Repebodies.

[0385]

[0395] Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block binding of the antigen binding domain to its cognate receptor. Advantageously, such moieties can also function as half-life extending elements. For example, a peptide that is modified by conjugation to a water-soluble polymer, such as PEG, can sterically inhibit or prevent binding of the cytokine to its receptor. Polypeptides, or fragments thereof, that have long serum half-lives can also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin and the like, as well as fragments and muteins of such polypeptides. Similarly, proteins or polypeptides, such as antibodies of antibody fragments, which bind such proteins, such as an scFv or single variable domain that binds human serum albumin can be used as blocking elements.

[0386]

[0396] Also disclosed herein are modified ABs that contain two or more blocking elements having specificity for the antigen binding domain and further contains a separate half-life extension element.

[0387]

[0397] A preferred blocking element is a peptide that contains an epitope for a binding domain. Such peptides can comprise a conformational or linear epitope that is specifically bound by a binding domain. Peptides that comprise linear epitopes typically comprise at least about 8 amino acids, and frequently between about 8 amino acids and about 20 amino acids but can be longer if desired. Peptides blockers can comprise the amino acid sequence of the immunogen used to create or isolate a binding domain. Peptide blockers can comprise an amino acid sequence that is the same as a region of the cognate antigen of the binding domain. Peptide blockers can also be any peptide that has binding affinity and specificity for the binding domain, such as engineered and synthetic peptides. In one example, a peptide that comprises the epitope for a binding domain is used to prepare a library of variant peptides. A peptide that binds to the binding domain with desired affinity can be identified and isolated from the library using suitable methods. In another example, peptides of random sequence can be designed or prepared and screed (e.g., in silico) to identify peptides that bind to a particular binding domain.

[0388]

[0398] A preferred blocking element is a peptide that contains an epitope for CD3epsilon. A preferred blocking element is a peptide that contains an epitope having SEQ ID NO: 496. A preferred blocking element is a peptide that contains an epitope having SEQ ID NO: 497. Such peptides can comprise a conformational or linear epitope that is specifically bound by a binding domain. Peptides that comprise linear epitopes typically comprise at least about 8 amino acids, and frequently between about 8 amino acids and about 20 amino acids but can be longer if desired.

[0389]

[0399] The blocking element can comprise a peptide that comprises or consists of the amino acid sequence of SEQ ID NO: 489, or an amino acid sequence that has at least 90% identity to SEQ ID NO:

[0390] 489.

[0391]

[0400] The blocking element can comprise a peptide that comprises or consists of the amino acid sequence of SEQ ID NO: 490. or an amino acid sequence that has at least 90% identity to SEQ ID NO:

[0392] 490.

[0393]

[0401] The blocking element can comprise a peptide that comprises or consists of the amino acid sequence of SEQ ID NO: 491, or an amino acid sequence that has at least 90% identity to SEQ ID NO:

[0394] 491.

[0395]

[0402] The blocking element can comprise a peptide that comprises or consists of the amino acid sequence of SEQ ID NO: 492. or an amino acid sequence that has at least 90% identity to SEQ ID NO:

[0396] 492.

[0397]

[0403] The blocking element can comprise a sequence that comprises or consists of the amino acid sequence of SEQ ID NO: 493, or an amino acid sequence that has at least 90% identity to SEQ ID NO:

[0398] 493.

[0399] F. Half-Life Extension Element

[0400]

[0404] The half-life extension element increases the in vivo half-life and provides altered pharmacodynamics and pharmacokinetics of tire modified ABs or immunoglobulin formats described herein. Without being bound by theory, the half-life extension element alters pharmacodynamic properties including alteration of tissue distribution, penetration, and diffusion of the modified Abs or immunoglobulin formats described herein. In some embodiments, the half-life extension element can improve tissue targeting, tissue penetration, diffusion within the tissue, and enhanced efficacy as compared with a protein without a half-life extension element. Without being bound by theory, an exemplary way to improve the pharmacokinetics of a polypeptide is by expression of an element in the polypeptide chain that binds to receptors that are recycled to the plasma membrane of cells rather than degraded in the lysosomes, such as the FcRn receptor on endothelial cells and transferrin receptor. Three types of proteins, e.g., human IgGs, HSA (or fragments), and transferrin, persist for much longer in human serum than would be predicted just by their size, which is a function of their ability to bind to receptors that are recycled rather than degraded in the lysosome. These proteins, or fragments that retain FcRn binding, are routinely linked to other polypeptides to extend their serum half-life. HSA may also be directly bound to the pharmaceutical compositions or bound via a short linker. Fragments of HSA may also be used. HSA and fragments thereof can function as both a blocking element and a half-life extension element. Human IgGs and Fc fragments can also cany out a similar function.

[0401]

[0405] Tire serum half-life extension element can also be an antigen-binding polypeptide that binds to a protein with a long serum half-life such as serum albumin (e.g., HSA), transferrin and the like. Examples of such polypeptides include antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a single chain variable fragment (scFv), an antigen binding fragment (Fab), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic antibody binding and / or structure such as, anticalins, Adnectin™, iMab, Anticalin®, EETI-II / AGRP, Kunitz domain, thioredoxin peptide aptamer, Affibody®, DARPin, Affilin, Tetranectin, Fynomer, and Avimer. Affimers. Alphabodies, Atrimers. Avimers, Centyrins. Obodies. Pronectins. and Repebodies. Further examples of antigen-binding polypeptides include a ligand for a desired receptor, a ligand-binding portion of a receptor, a lectin, and peptides that bind to or associate with one or more target antigens. The antibodies and fragments thereof can function as both a blocking element and a half-life extension element.

[0402]

[0406] The half-life extension element can also function as both a blocking element and a half-life extension element. For instance, the half-life extension element (e.g., HSA binding polypeptide) can function as a blocking element (that sterically inhibits binding of the antigen binding domain to its target antigen).

[0403]

[0407] Tire half-life extension element as provided herein is preferably a human serum albumin (HSA), an antigen binding polypeptide that binds human serum albumin, or an immunoglobulin Fc or fragment thereof. The half-life extension element can comprise an Fc domain or a fragment thereof. For example, the Fc domain can comprise a CH2 and CH3 domain or fragment thereof. For example, the Fc domain can comprise a constant domain of the heavy chain polypeptide. Typically, the half-life extension element is an antibody or fragment thereof (particularly a dAb), e.g., an antibody or fragment that binds serum albumin (particularly HSA), an immunoglobulin Fc or fragment thereof, or serum albumin (particularly HSA).

[0404]

[0408] Preferably, the half-life extension element can be a single domain antibody fragment (sdAb).

[0405]

[0409] sdAbs disclosed herein can compete with FcRn (human FcRn) for binding to human serum albumin and, preferably, bind to domain 1 (DI) of human serum albumin. Contrary to the well-accepted knowledge in the art that anti-albumin binding domains that interfere with albumin binding to FcRn are not suitable for half-life extension applications, the inventors have discovered that sdAbs that compete with FcRn (human FcRn) for binding to human serum albumin can effectively extend the half-life (e.g., the in vivo serum half-life) of short half-life therapeutic agents (e.g., prodrug polypeptides).

[0406]

[0410] The sdAbs of this disclosure include, but are not limited to, heavy chain variable domain (VH). variable heavy domain of heavy chain (VHH), single domain shark variable domain of new antigen receptor (VNAR), or light chain variable (VL) domain (e.g., a kappa VL domain or a lambda VL domain). [4H] Tire sdAbs comprise one or more complementary determining regions (CDRs), typically three CDRs, of an immunoglobulin single variable domain that has binding specificity for serum albumin. The CDRs of the sdAbs disclosed herein can be derived from a camelid. Hie sdAbs can be humanized and comprise one or more FRs, that can be derived from a human germline and typically include back mutations. The sdAbs disclosed herein can comprise a CDR1, a CDR2 and / or a CDR3. Preferably, the sdAbs disclosed herein comprise CDR1, CDR2, and CDR3.

[0407]

[0412] The sdAbs can comprise a CDR1 comprising an amino acid sequence of Formula (I):

[0408] Xi X2X3X4X5X6AX7X8G (I) .

[0409]

[0413] In Fonnula (I), Xi is glycine or aspartic acid, X2is glycine, arginine, phenylalanine, leucine, valine, serine, tyrosine, histidine, or threonine, X3is threonine, glycine, or serine, X4is phenylalanine, valine, tyrosine, leucine, isoleucine, or arginine, X5is aspartic acid, serine, threonine, or arginine, X6is aspartic acid, serine, glycine, or glutamic acid, X7is arginine, glycine, serine, threonine, methionine, valine, isoleucine, histidine, or asparagine and X8is arginine, glycine, leucine, methionine, isoleucine or valine.

[0410]

[0414] Tire sdAbs can comprise a CDR2 comprising an amino acid of Formula (II)

[0411] A l S Xi S X2X3 X4T X5 YX6X7X8 V K G (II) (SEQ ID NO: 918)

[0412]

[0415] In Fonnulas (II), Xi is isoleucine or alanine. X2is glycine or threonine, X3is glutamic acid or glycine. X4 is arginine, threonine, or serine, X5 is asparagine, arginine, or tyrosine. Xe is glycine, arginine, serine, or glutamic acid, X7is glutamic acid, lysine, aspartic acid, or glutamic acid, and X8is serine or glutamic acid.

[0413]

[0416] Tire sdAbs can comprise a CDR3 comprising an amino acid sequence of Formula (III): A Xi G D W Y H L X2Q X3X4X5X6X7X81 X9Xw(IIII) (SEQ ID NO: 919).

[0414]

[0417] In Fonnula (IV), Xi is serine or alanine, X2is valine, isoleucine, or threonine, X3 is glycine or glutamic acid, X4is threonine, glutamic acid, or histidine, X4is threonine, glutamic acid, or histidine, Xs is glycine or asparagine, X6us methionine or asparagine, X7is serine, alanine, or methionine, Xsis tyrosine or phenylalanine, X9is arginine or tryptophan, and Xiois asparagine or tyrosine.

[0415]

[0418] In embodiments, the sdAbs, comprises a CDR1 of Formula (I), a CDR2 of Formula (II), and a CDR3 of Formula (III).

[0416]

[0419] The modified AB’s or immunoglobulin formats (e.g., prodrug polypeptides) described herein can comprise a sdAbs that comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, 8, 37 or a variant of any of the foregoing as described herein. The sdAbs disclosed herein can include a CDR1 that comprises or consists of SEQ ID NO: 1. The sdAbs disclosed herein can include a CDR1 that comprises or consists of SEQ ID NO: 4. Tire sdAbs disclosed herein can include a CDR1 that comprises or consists of SEQ ID NO: 7. Tire sdAbs disclosed herein can comprise a CDR1 that comprises or consists of SEQ ID NO: 8. The sdAbs disclosed herein can comprise a CDR1 that comprises or consists of SEQ ID NO: 37.

[0417]

[0420] The modified AB’s or immunoglobulin formats described herein can comprise a sdAbs that comprises a CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 2, 5, 9, 10, 11, 38 or a variant of any of the foregoing as described herein. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 2. Tire sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 5. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 9. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 10. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 11. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 38.

[0418]

[0421] Tire modified AB's or immunoglobulin formats described herein can comprise a sdAb that comprises a CDR3 as defined by any one of SEQ ID NOs: 3, 6, or 12, or a variant of any of the foregoing as described herein. The sdAbs disclosed herein can include a CDR3 that comprises or consists of SEQ ID NO: 3. The sdAbs disclosed herein can include a CDR3 that comprises or consists of SEQ ID NO: 6. The sdAbs disclosed herein can include a CDR3 that comprises or consists of SEQ ID NO: 12.

[0419]

[0422] Tire modified AB’s or immunoglobulin formats described herein can comprise a sdAb that comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, 8, 37, or a variant of any of the foregoing, a CDR2 comprising any one of SEQ ID NO: 2, 5, 9. 10, 11, 38, or a variant of any of the foregoing, and a CDR3, such that the sdAbs binds human serum albumin.

[0420]

[0423] The modified AB’s or immunoglobulin formats described herein can comprise a sdAb that comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 1, 4, 7, 8, 37, or a variant of any of tire foregoing, a CDR3 comprising any one of SEQ ID NO: 3, 6, or 12, or a variant of any of the foregoing, and a CDR2, such that the sdAbs binds human serum albumin.

[0421]

[0424] The modified AB’s or immunoglobulin formats described herein can comprise a sdAb that comprises a CDR2 comprising any one of SEQ ID NO: 2, 5, 9, 10, 11, 38, or a variant of any of the foregoing, a CDR3 comprising any one of SEQ ID NO: 3, 6, or 12, or a variant of any of the foregoing, and a CDR1, such that the sdAbs binds human serum albumin.

[0422]

[0425] Typically, modified AB's or immunoglobulin formats described herein comprise sdAbs that have all three CDRs (e.g.. a CDR1, a CDR2, and a CDR3).

[0423]

[0426] The sdAbs can comprise a CDR1 comprising any one of SEQ ID NO: 1, 4, 7, 8 and 37, or a variant of any of the foregoing; a CDR2 comprising any one of SEQ ID NO: 2, 5, 9, 10, 11 and 38 or a variant of any of the foregoing; and a CDR3 comprising any one of SEQ ID NO: 3, 6, and 12, or a variant of any of the foregoing.

[0424]

[0427] The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 1 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 2 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 3 or variant thereof. The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 1, CDR2 that comprises or consists of SEQ ID NO: 2, and CDR3 that comprises or consists of SEQ ID NO: 3.

[0428] The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 4 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 5 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 6 or variant thereof. The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 4, CDR2 that comprises or consists of SEQ ID NO: 5, and CDR3 that comprises or consists of SEQ ID NO: 6.

[0425]

[0429] The sdAbs can comprise CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof. CDR1 that comprises or consists of SEQ ID NO: 7, 37 or 8 or variant of any of the foregoing, and CDR2 that comprises or consists of SEQ ID NO: 38, 9, 10 or 11 or variant of any of the foregoing. The sdAbs can comprise CDR3 that comprises or consists of SEQ ID NO: 12, CDR1 that comprises or consists of SEQ ID NO: 7, 37 or 8, and CDR2 that comprises or consists of SEQ ID NO: 38, 9, 10 or 11.

[0426]

[0430] The modified AB's or immunoglobulin formats described herein can comprise an sdAb comprising a) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof. CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; b) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; c) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; d) CDR1 that comprises or consists of SEQ ID NO: 7. CDR2 that comprises or consists of SEQ ID NO: 10. and CDR3 that comprises or consists of SEQ ID NO: 12; e) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; f) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; g) CDR1 that comprises or consists of SEQ ID NO: 7 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; h) CDR1 that comprises or consists of SEQ ID NO: 7, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; i) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; j) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 9. and CDR3 that comprises or consists of SEQ ID NO: 12; k) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; 1) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 10, and CDR3 that comprises or consists of SEQ ID NO: 12; m) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; n) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; o) CDR1 that comprises or consists of SEQ ID NO: 37 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; p) CDR1 that comprises or consists of SEQ ID NO: 37, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12; q) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, a CDR2 that comprises or consists of SEQ ID NO: 9 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; r) CDR1 that comprises or consists of SEQ ID NO: 8, a CDR2 that comprises or consists of SEQ ID NO: 9, and CDR3 that comprises or consists of SEQ ID NO: 12; s) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 10 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; t) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 10. and CDR3 that comprises or consists of SEQ ID NO: 12; u) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 11 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; v) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 11, and CDR3 that comprises or consists of SEQ ID NO: 12; w) CDR1 that comprises or consists of SEQ ID NO: 8 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 12 or variant thereof; x) CDR1 that comprises or consists of SEQ ID NO: 8, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 12.

[0427]

[0431] The CDRs of the sdAbs disclosed herein are interposed between stretches of amino acids referred to as framework regions (FRs). The sdAbs disclosed herein can comprise FR1, FR2, FR3, and / or F4. In certain embodiments, the FRs regions of the sdAbs disclosed herein comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more of amino acid sequence homology to human FRs. Typically, the FRs can be derived from a human germline gene. Exemplary human germline genes include, but are not limited to, IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16, IGHV3-19, IGHV3-20, IGHV3-21, IGHV3-22, IGHV3-23, IGHV3- 25, IGHV3-29, IGHV3-30, IGHV3-32, IGHV3-33, IGHV3-35, IGHV3-36, IGHV3-37, IGHV3-38, IGHV3-41, IGHV3-42, IGHV3-43, IGHV3-47, IGHV3-38, IGHV3-49, IGHV3-50, IGHV3-52, IGHV3- 53, IGHV3-54, IGHV3-57, IGHV3-60, IGHV3-62, IGHV3-63, IGHV3-64, IGHV3-66, IGHV3-69, IGHV3-71, IGHV3-72, IGHV3-73, IGHV3-74, IGHV3-75, IGHV3-76, or IGHV3-79. Certain preferred sdAbs include FRs derived from human germline gene IGHV3-11 and / or IGHV3-23.

[0428]

[0432] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 13, 14, 31, or a variant of any of the foregoing. In some instances, the sdAbs disclosed herein can comprise a FR1 comprising an amino acid sequence that has at least 80% identity to any one of SEQ ID NOs.: SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing. The sdAbs disclosed herein can include a FR1 that comprises or consists of SEQ ID NO: 13. The sdAbs disclosed herein can include a FR1 that comprises or consists of SEQ ID NO: 14. The sdAbs disclosed herein can include a FR1 that comprises or consists of SEQ ID NO: 31.

[0429]

[0433] The sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 15-19, 24-28, 32, 33, or a variant of any of the foregoing. In some instances, the sdAbs disclosed herein can comprise a FR2 comprising an amino acid sequence that has at least 80% identity to any one of SEQ ID NOs: 15-19, 24-28, 32. 33, or a variant of any of the foregoing. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 15. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 16. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 17. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 18. Tire sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 19. Tire sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 24. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 25. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 26. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 27. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 28. Tire sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 32. Tire sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 33.

[0430]

[0434] The sdAbs disclosed herein can comprise a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing. In some instances, the sdAbs disclosed herein can comprise a FR2 comprising an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 20. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 21. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 34. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 35. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 36.

[0431]

[0435] The sdAbs disclosed herein can comprise a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof. In some instances, the sdAbs disclosed herein can comprise a FR4 comprising an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof. Tire sdAbs disclosed herein can include a FR4 that comprises or consists of SEQ ID NO: 22.

[0432]

[0436] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; and a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 15-19, 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 15- 19, 24-28, 32, 33, or a variant of any of the foregoing.

[0433]

[0437] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; and a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35. or 36, or a variant of any of the foregoing.

[0434]

[0438] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0435]

[0439] The sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 15-19, 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 15-19. 24-28, 32, 33, or a variant of any of the foregoing; and a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing.

[0436]

[0440] Tire sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 15-19, 24-28, 32, 33, or a variant of any of tire foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 15-19. 24-28, 32, 33, or a variant of any of tire foregoing: and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0437]

[0441] The sdAbs disclosed herein can comprise a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22. or a variant thereof.

[0438]

[0442] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 15-19. 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 15-19, 24- 28, 32, 33. or a variant of any of the foregoing; and a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing.

[0439]

[0443] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13. 14, 31, or a variant of any of the foregoing; a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 15-19, 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 15-19, 24- 28, 32, 33, or a variant of any of tire foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0440]

[0444] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0441]

[0445] Tire sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 15-19, 24-28, 32, 33, or a variant of any of tire foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 15-19, 24-28, 32, 33, or a variant of any of tire foregoing; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0442]

[0446] Typically, the sdAbs disclosed herein have all four FRs (e g., a FR1, a FR2, a FR3, and a FR4). The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 13, 14, 31, or a variant of any of the foregoing; a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 15-19, 24-28, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 15-19, 24-28, 32, 33, or a variant of any of the foregoing; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 20, 21, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 22, or a variant thereof.

[0443]

[0447] Tire sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 15, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0444]

[0448] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 16, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0449] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 17, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0445]

[0450] Tire sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 18, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0446]

[0451] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 15, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0447]

[0452] Tire sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 16, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0448]

[0453] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 17, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0449]

[0454] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 18, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0450]

[0455] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 19, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0451]

[0456] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 24, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0452]

[0457] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 25, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0453]

[0458] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 26, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0459] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 27, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0454]

[0460] Tire sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 24, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0455]

[0461] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 25, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0456]

[0462] Tire sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 26, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0457]

[0463] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 27, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0458]

[0464] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 28, a FR3 that comprises or consists of SEQ ID NO: 21; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0459]

[0465] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 14, a FR2 that comprises or consists of SEQ ID NO: 24, a FR3 that comprises or consists of SEQ ID NO: 30; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0460]

[0466] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 32, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0461]

[0467] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 33, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0462]

[0468] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 27, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0469] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 13, a FR2 that comprises or consists of SEQ ID NO: 25, a FR3 that comprises or consists of SEQ ID NO: 20; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0463]

[0470] Tire sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 32, a FR3 that comprises or consists of SEQ ID NO: 34; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0464]

[0471] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 33, a FR3 that comprises or consists of SEQ ID NO: 34; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0465]

[0472] Tire sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 27, a FR3 that comprises or consists of SEQ ID NO: 34; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0466]

[0473] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 25, a FR3 that comprises or consists of SEQ ID NO: 34; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0467]

[0474] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 32, a FR3 that comprises or consists of SEQ ID NO: 35; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0468]

[0475] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 33, a FR3 that comprises or consists of SEQ ID NO: 35; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0469]

[0476] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 27, a FR3 that comprises or consists of SEQ ID NO: 35; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0470]

[0477] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 25, a FR3 that comprises or consists of SEQ ID NO: 35; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0471]

[0478] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 31, a FR2 that comprises or consists of SEQ ID NO: 28, a FR3 that comprises or consists of SEQ ID NO: 36; and a FR4 that comprises or consists of SEQ ID NO: 22.

[0479] The modified AB’s or immunoglobulin formats described herein disclosed herein can comprise or consist of any one of the sdAbs disclosed in Table 2.

[0472] Table 2. Exemplary sdAbs

[0473]

[0480] Tire modified AB’s or immunoglobulin formats (e.g., prodrug polypeptides) described herein can comprise an sdAb comprising or consisting of tire amino acid sequence of any of SEQ ID NOs: 39-79 or 426-430, or an amino acid sequence that has at least about 80% identity to any of SEQ ID NOs: 39-79 or 426-430. For example, tire sdAbs can comprise or consist of an amino acid sequence that has at least about 81% identity, at least about 82% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to any of SEQ ID NOs: 39, 40, 41, 42. 43. 44. 45, 46, 47, 48, 49, 50, 51. 52. 53, 54, 55, 56, 57, 58, 59, 60. 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 426, 427, 428, 429, 430.

[0474]

[0481] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 39.

[0475]

[0482] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 40.

[0476]

[0483] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 41.

[0477]

[0484] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 42.

[0478]

[0485] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 43.

[0486] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 44. The sdAb

[0479]

[0487] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 45.

[0480]

[0488] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 46.

[0481]

[0489] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 47.

[0482]

[0490] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 48.

[0483]

[0491] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 49.

[0484]

[0492] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 50.

[0485]

[0493] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 51.

[0486]

[0494] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 52.

[0487]

[0495] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 53.

[0488]

[0496] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 54.

[0489]

[0497] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 55, , or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 55.

[0490]

[0498] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 56.

[0491]

[0499] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 57, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 57.

[0492]

[0500] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 58.

[0501] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 59.

[0493]

[0502] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 60.

[0494]

[0503] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 61.

[0495]

[0504] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 62.

[0496]

[0505] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 63.

[0497]

[0506] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 64.

[0498]

[0507] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 65.

[0499]

[0508] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 66.

[0500]

[0509] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 67.

[0501]

[0510] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 68.

[0502] [5H] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 69.

[0503]

[0512] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 70.

[0504]

[0513] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 71.

[0505]

[0514] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 72.

[0506]

[0515] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 73.

[0516] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 74.

[0507]

[0517] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 75.

[0508]

[0518] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 76. , or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 76.

[0509]

[0519] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 77.

[0510]

[0520] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 79.

[0511]

[0521] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 79.

[0512]

[0522] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 426, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 426.

[0513]

[0523] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 427, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 427.

[0514]

[0524] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 428, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 428.

[0515]

[0525] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 429, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 429.

[0516]

[0526] Tire sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 430, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 430.

[0517]

[0527] The sdAb can comprise or consist of SEQ ID NO: 494, or a variant thereof. Tire sdAB can comprise or consist of the amino acid sequence of SEQ ID NO: 494, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 494.

[0518]

[0528] The sdAbs disclosed herein include functional variants that differ in amino acid sequence in comparison, for example, to any one of SEQ ID NOs.: 39-79 or 426-430. A “functional variant” of a sdAb retains the ability to bind to serum albumin.

[0519]

[0529] The sdAbs disclosed herein can comprise one or more amino acid substitutions. Tire amino acid substitution can be a conservative substitution or a non-conservative substitution, but preferably is a conservative substitution. Conservative amino acid substitutions are generally made in accordance with the following Table 3.

[0520] Table 3. Exemplary amino acid substitutions

[0521]

[0530] As described herein, a sdAb of this disclosure can contain a CDR1 that comprises or consists of

[0522] SEQ ID NO: 1, 4, 7, 8, or 37 or a variant of any of the forgoing. A variant of SEQ ID NO: 1, 4, 7, 8, or 37 has at least about 80% or at least about 90% amino acid sequence identity to SEQ ID NO: 1, 4, 7, 8, or 37; a variant of SEQ ID NO: 1, 4, 7, 8 or 37 can contain one or two amino acid substitutions. As amino acid substitutions in the CDRs (i.e., CDR1, CDR2 and / or CDR3) can affect binding to serum albumin, it is preferred that any such substitutions are conservative substitutions.

[0523]

[0531] As described herein, a sdAb of this disclosure can contain a CDR2 that comprises or consists of SEQ ID NO: 2, 5, 9. 10. 11 or 38 or a variant of any of the forgoing. A variant of SEQ ID NO: 2, 5, 9. 10, 11 or 38 has at least about 80%, at least about 85%, or at least about 90% amino acid sequence identity to SEQ ID NO: 2, 5, 9, 10, 11 or 38; a variant of SEQ ID NO: 2, 5, 9, 10, 11 or 38 can contain one or two or three amino acid substitutions.

[0524]

[0532] As described herein, a sdAb of this disclosure can contain a CDR3 that comprises or consists of SEQ ID NO: 3, 6 or 12 or a variant of any of the forgoing. A variant of SEQ ID NO: 3, 6 or 12 has at least about 80%, at least about 85% or at least about 90% amino acid sequence identity to SEQ ID NO: 3, 6 or 12; a variant of SEQ ID NO: 3, 6 or 12 can contain one or two or three amino acid substitutions.

[0525]

[0533] As described herein, a sdAb of this disclosure can contain FRs and these sequences can generally include amino acid substitutions without causing a loss of binding activity. As described herein, a sdAb of this disclosure can contain a FR1 that comprises or consists of SEQ ID NO: 13, 14, or 31 or a variant of any of the forgoing. A variant of SEQ ID NO: 3. 6 or 12 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 3. 6 or 12.

[0526]

[0534] As described herein, a sdAb of this disclosure can contain a FR2 that comprises or consists of SEQ ID NO: 15-19, 24-28, 32, or 33 or a variant of any of tire forgoing. A variant of SEQ ID NO: 15-19, 24-28, 32, or 33 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 15-19. 24-28, 32, or 33.

[0527]

[0535] As described herein, a sdAb of this disclosure can contain a FR3 that comprises or consists of SEQ ID NO: 20, 21. 29. 30. 34, 35, or 36 or a variant of any of the forgoing. A variant of SEQ ID NO: 20. 21, 29, 30, 34, 35, or 36 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 20, 21, 29, 30, 34, 35, or 36.

[0528]

[0536] As described herein, a sdAb of this disclosure can contain a FR4 that comprises or consists of SEQ ID NO: 22 or a variant thereof. A variant of SEQ ID NO: 22 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 22.

[0529] The sdAb disclosed herein comprising the amino acid sequence of any one of SEQ ID NO: 39-79 or 426- 430 can comprises any one of the following amino acids: 1) the amino acid at residue at position 24 can be selected from alanine or serine; 2) the amino acid at position 44 can be selected from glutamic acid, glycine, or aspartic acid; 3) the amino acid at position 45 can be selected from arginine, or leucine; 4) the amino acid at position 78 can be selected from asparagine, serine, threonine, or leucine; or 5) the amino acid at position 79 can be selected from valine, leucine, or phenylalanine

[0530] G. Cleavable Linkers

[0531]

[0537] As disclosed herein, the modified AB’s or immunoglobulin formats described herein comprises one or more linker sequences. A linker sequence serves to provide flexibility between the polypeptides, such that, for example, the blocking domain is capable of inhibiting the activity of the antigen binding domain. The linker can be located between the antibody binding domain, the blocking element and / or the half-life extension element, when present.

[0532]

[0538] As described herein the modified ABs and / or immunoglobulin formats comprises a protease cleavable linker. The protease cleavable linker can comprise one or more cleavage sites for one or more desired protease. Preferably, the desired protease is enriched or selectively expressed at the desired target site (e.g., the tumor microenvironment or site of inflammation). Thus, the modified antibodies or antibody binding fragments is preferentially or selectively cleaved at the target site.

[0533]

[0539] Suitable linkers are typically less than about 100 amino acids. Such linkers can be of different lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino acids, from 1 amino acid to 60 amino acids, from 1 to 70 amino acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from 1 to 100 amino acids. In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 83, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99m or about 100 amino acids in length. Preferred linkers are typically from about 5 amino acids to about 30 amino acids.

[0534]

[0540] Preferably the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked amino acid sequence of interest. In a preferred embodiment, the linker is cleavable by a cleaving agent, e.g., an enzyme. Preferably, the linker comprises a protease cleavage site. In some cases, the linker comprises one or more cleavage sites. The linker can comprise a single protease cleavage site. The linker can also comprise 2 or more protease cleavage sites. For example, 2 cleavage sites, 3 cleavage sites, 4, cleavage sites, 5 cleavage sites, or more. In cases where the linker comprises 2 or more protease cleavage sites, the cleavage sites can be cleaved by tire same protease or different proteases. A linker comprising two or more cleavage sites is referred to as a "‘tandem linker." The two or more cleavage sites can be arranged in any desired orientation, including, but not limited tom one cleavage site adjacent to another cleavage site, one cleavage site overlapping another cleavage site, or one cleavage site following by another cleavage site with intervening amino acids between the two cleavage sites.

[0535]

[0541] Tandem linkers can comprise two or more protease cleavable linkers described herein. For example, a tandem linker can comprise SEQ ID NO: 707 and SEQ ID NO: 708. For example, a tandem linker can comprise SEQ ID NO: 514 and SEQ ID NO: 707. For example, a tandem linker can comprise SEQ ID NO: 514 and SEQ ID NO: 708. For example, a tandem linker can comprise SEQ ID NO: 513 and SEQ ID NO: 514. For example, a tandem linker can comprise SEQ ID NO: 513 and SEQ ID NO: 707. For example, a tandem linker can comprise SEQ ID NO: 513 and SEQ ID NO: 708. The tandem linkers can further comprise a non-cleavable amino acid sequence between the protease cleavable linkers. As an example, the linker can comprise sggpALFKSSFPpgsGGGGSGGGGSsggpTDFALQAVSApgs (SEQ ID NO: 946)

[0536]

[0542] Of particular interest in the present invention are disease specific protease-cleavable linkers. Also preferred are protease-cleavable linkers that are preferentially cleaved at a desired location in the body, such as the tumor microenvironment, relative to the peripheral circulation.

[0537]

[0543] Proteases known to be associated with diseased cells or tissues include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E. Cathepsin G. Cathepsin K. Cathepsin L, kallikreins, hKL hK10, hK15. plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin- like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin- 10 converting enzyme, thrombin, FAP (FAPa), dipeptidyl peptidase, meprins, granzymes and dipeptidyl peptidase IV (DPPIV / CD26). Proteases capable of cleaving linker amino acid sequences (which can be encoded by the chimeric nucleic acid sequences provided herein) can, for example, be selected from the group consisting of a prostate specific antigen (PSA), a matrix metalloproteinase (MMP), an A Disintigrin and a Metalloproteinase (ADAM), a plasminogen activator, a cathepsin, a caspase, a tumor cell surface protease, and an elastase. The MMP can, for example, be matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 14 (MMP 14). In addition, or alternatively, the linker can be cleaved by a cathepsin, such as, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K and / or Cathepsin L. Preferably, the linker can be cleaved by MMP 14 or Cathepsin L.

[0538]

[0544] Proteases useful for cleavage of linkers and for use in the modified ABs disclosed herein, including immunoglobulin formats disclosed herein are presented in Table 4, and exemplar.- proteases and their cleavage site are presented in Table 5.

[0539] Table 4. Proteases relevant to inflammation and cancer

[0540] Table 5. Exemplary Proteases and Protease Recognition Sequences

[0541]

[0545] Exemplary protease cleavable linkers include, but are not limited to kallikrein cleavable linkers, thrombin cleavable linkers, chymase cleavable linkers, carboxypeptidase A cleavable linkers, cathepsin cleavable linkers, elastase cleavable linkers. FAP cleavable linkers, ADAM cleavable linkers, PR-3 cleavable linkers, granzyme M cleavable linkers, a calpain cleavable linkers, a matrix metalloproteinase (MMP) cleavable linkers, a plasminogen activator cleavable linkers, a caspase cleavable linkers, a tryptasc cleavable linkers, or a tumor cell surface protease. Specifically, MMP9 cleavable linkers, ADAM cleavable linkers, CTSL1 cleavable linkers, FAPa cleavable linkers, and cathepsin cleavable linkers. Some preferred protease-cleavable linkers are cleaved by a MMP and / or a cathepsin.

[0542]

[0001] Exemplary linkers that may be suitable for the modified antibodies or antibody binding fragments disclosed herein are disclosed in International Publication No.: W02020 / 232305. For example, the linker can comprise the sequence GPAGLYAQ (SEQ ID NO: 510); GPAGMKGL (SEQ ID NO: 511); PGGPAGIG (SEQ ID NO: 512); ALFKSSFP (SEQ ID NO: 513); ALFFSSPP (SEQ ID NO: 514); LAQRLRSS (SEQ ID NO: 515); LAQKLKSS (SEQ ID NO: 516);

[0543] GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (SEQ ID NO: 517); RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (SEQ ID NO: 518); KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR (SEQ ID NO: 519); RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (SEQ ID NO: 520); KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (SEQ ID NO: 521); SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (SEQ ID NO: 522); RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (SEQ ID NO: 523); GGGALFKSSFPLAQKLKSSPGGPAGIGGGR (SEQ ID NO: 524); RGPGGPAGIGPLAQKLKSSALFKSSFPGGG (SEQ ID NO: 525); RGGPLAQKLKSSPGGPAGIGALFKSSFPGK (SEQ ID NO: 526); RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (SEQ ID NO: 527); GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (SEQ ID NO: 528); GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (SEQ ID NO: 529); RGGALFKSSFPLAQKLKSSGPAGLYAQGGK (SEQ ID NO: 530); RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (SEQ ID NO: 531); SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (SEQ ID NO: 532); KGGPGGPAGIGPLAQRLRSSALFKSSFPGR (SEQ ID NO: 533); KSGPGGPAGIGALFFSSPPLAQKLKSSGGR (SEQ ID NO: 534); or SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG (SEQ ID NO: 535)

[0544]

[0546] Other exemplary- linkers that may be suitable for the modified ABs or immunoglobulin formats described herein can comprise the sequence TDFALQAVSA (SEQ ID NO: 707) or ALFTSIAF (SEQ ID NO: 708).

[0545]

[0547] Other exemplary linkers that may be suitable for the modified ABs or immunoglobulin formats described herein can comprise the sequence PLAQKLKSS (SEQ ID NO: 942), GPAGLYAQPLAQKLKSS (SEQ ID NO: 943), ALLKSIFP (SEQ ID NO: 1011), PLAQKVKSSFP (SEQ ID NO: 944), PLAQKLRSSFP (SEQ ID NO: 945) and sggpALFKSSFPpgsGGGGSGGGGSsggpTDFALQAVSApgs (SEQ ID NO: 946).

[0548] Certain preferred modified ABs or immunoglobulin formats comprise the sequence GPAGLYAQ (SEQ ID NO: 510) or ALFKSSFP (SEQ ID NO: 513). Certain preferred ABs or immunoglobulin formats comprise the sequence TDFALQAVSA (SEQ ID NO: 707) or ALFTSIAF (SEQ ID NO: 708). Certain preferred modified ABs or immunoglobulin formats comprise sggpALFKSSFPpgsGGGGSGGGGSsggpTDFALQAVSApgs (SEQ ID NO: 946)

[0546]

[0549] Preferred linkers comprising more than one cleavage motif comprise the amino acids selected from SEQ ID NOs: 510 to 535. In some embodiments, the linker comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% identical to SEQ ID NOs: 510 to 535.

[0547]

[0550] Tire linker can comprise one or more cleavage motif or functional variants that are the same or different. The linker can comprise 1, 2, 3, 4, 5, or more cleavage motifs or functional variants. Linker comprising 30 amino acids can contain 2 cleavage motifs or functional variants. 3 cleavage motifs or functional variants or more. A '‘functional variant” of a linker retains the ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease) and are not cleaved or cleaved with low efficiency in the periphery (e.g., serum). For example, the functional variants retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a linker comprising any one of SEQ ID NOs: 510 to 535.

[0548]

[0551] The length, degree of flexibility and other properties of the linkers used in the modified antibodies or antibody binding fragments may have some influence on properties, including, but not limited to the affinity, the specificity, or avidity for one or more components. In some instances, the linker can comprise flexible residues (such as glycine or serine) so that the adjacent amino acid sequence of interest is free (e.g., blocking element or antibody binding domain) to move relative to each other.

[0549]

[0552] Other linker considerations include, but are not limited to. the effect on physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability, rigidity, flexibility, immunogenicity, modulation of antibody binding, the ability to be incorporated into a micelle or liposome.

[0550]

[0553] In some instances, a non-peptide linker may be suitable. For example, such linkage can be through covalent binding, affinity binding, intercalation, coordinate binding, and complexation. Covalent linking can be achieved by direct condensation of existing side chains or by the incorporation of external bridging molecules. Representative coupling agents include organic compounds such as thioesters, carbodiimide, succinimide esters, diisocyanate, glutaraldehyde, diazobenzenes, and hexamethylene

[0551] I ll diamines. Non-peptide linkers are well established in the art and may be suitable for the modified antibodies or antibody binding fragments described herein.

[0552]

[0554] Tire linker desirably remains stable in the circulation for at least 2 hours, at least 5, hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or longer.

[0553]

[0555] In some embodiments, the linker is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% in the circulation as compared to the target location. The linker is also stable in the absence of an enzyme capable of cleaving the linker. However, upon expose to a suitable enzyme (i.e., a protease), the linker is cleaved resulting in separation of the linked domain.

[0554]

[0556] The modified Abs and / or immunoglobulin fonnats can comprise one or more flexible linkers. Flexible linkers can be included to provide tire desired flexibility in the modified AB and / or immunoglobulin formats. The flexible linkers can be the same or different.

[0555]

[0557] When the modified AB and / or or immunoglobulin formats includes a flexible linker, it can comprise or consist of tire amino acid sequence of SEQ ID NO: 495.

[0556]

[0558] When the modified ABs or immunoglobulin fonnats includes a flexible linker, it can comprise or consist of the sequence GGGGS (SEQ ID NO: 920), or GGGGSGGGGSGGGGS (SEQ ID NO: 924).

[0557]

[0559] The first antigen binding domain can be operably linked to the target antigen binding domain through one or more linkers as described herein. Suitable linkers will be apparent to those of skill in the art. Suitable linkers can include synthetic linkers, peptide linkers, and chemical bonds. Suitable linkers can include flexible linkers. Typically, the immunoglobulin formats described herein, such as T-cell engagers, use a peptide linker or a flexible linker. The peptide linker can be a short peptide linker or a long peptide linker. A short peptide linker can be about four amino acids to about 6 amino acids in length. For example, the short peptide linker can be 4 amino acids, 5 amino acids, or 6 amino acids. A long peptide linker can be about 10 amino acids to about 15 amino acids in length. For example, the short peptide linker can be 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, or 15 amino acids.

[0558]

[0560] Exemplary short peptide linkers and long peptide linkers are provided below in Table 35. Combinations of the peptide linkers that can be used to operably link the first antigen binding domain and target antigen binding domain are further provided in Table 35. Table 35.

[0559]

[0561] Exemplary flexible linkers are provided in Table 36. Combinations of flexible linkers that can be used to operably link the first antigen binding domain and target antigen binding domain are further provided in Table 36.

[0560] Table 36. GGGGS-type linkers

[0561] H. Exemplary T-cell Engagers

[0562] The T-cell engagers disclosed herein can also be referred to as prodrug polypeptide and / or multi-specific antibodies.

[0563]

[0562] As described herein, the T-cell engagers can comprise (i) a first antigen binding domain that has specificity for an antigen on a T-cell (e,g, CD3) (e.g., sdAbs, scFv, Fab etc.), (ii) preferably two or more blocking elements that have binding specificity for the first antigen binding domain that are operably linked through a cleavable linker to the first antigen binding domain, (iii) an optional half-life extension element, (iv) a target antigen binding domain (e.g., sdAbs, scFv, Fab etc.), and (v) at least one protease cleavable linker. The T-cell engager can further comprise one or more blocking elements having binding specificity for the target antigen domain. As described herein, the T-cell engagers can comprise two or more polypeptide chains.

[0564]

[0563] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g.. CD3) can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 999, a CDR2 having the amino acid sequence of SEQ ID NO: 1000, and a CDR3 having the amino acid of SEQ ID NO: 1001, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1002, a CDR2 having the amino acid sequence of SEQ ID NO: 1003, and a CDR3 having tire amino acid of SEQ ID NO: 1004.

[0565]

[0564] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1005, a CDR2 having the amino acid sequence of SEQ ID NO: 1006, and a CDR3 having the amino acid of SEQ ID NO: 1007, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1008, a CDR2 having the amino acid sequence of SEQ ID NO: 1009, and a CDR3 having tire amino acid of SEQ ID NO: 1010. The first antigen binding domain specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 997 and a light chain variable region having the amino acid sequence of SEQ ID NO: 998.

[0566]

[0565] The first antigen binding domain specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 995 and a light chain variable region having the amino acid sequence of SEQ ID NO: 996.

[0567]

[0566] The first antigen binding domain specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 997 and a light chain variable region having the amino acid sequence of SEQ ID NO: 998.

[0568]

[0567] The first antigen binding domain that has specificity7for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 863 and a light chain variable region comprising SEQ ID NO: 890.

[0569]

[0568] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g.. CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 864 and a light chain variable region comprising SEQ ID NO: 891.

[0570]

[0569] The first antigen binding domain that has specificity7for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 865 and a light chain variable region comprising SEQ ID NO: 892.

[0571]

[0570] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g.. CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 866 and a light chain variable region comprising SEQ ID NO: 893.

[0571] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 867 and a light chain variable region comprising SEQ ID NO: 894.

[0572]

[0572] Tire first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 868 and a light chain variable region comprising SEQ ID NO: 895.

[0573]

[0573] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 689 and a light chain variable region comprising SEQ ID NO: 896.

[0574]

[0574] Tire first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 870 and a light chain variable region comprising SEQ ID NO: 897.

[0575]

[0575] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 871 and a light chain variable region comprising SEQ ID NO: 898.

[0576]

[0576] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 872 and a light chain variable region comprising SEQ ID NO: 899.

[0577]

[0577] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 873 and a light chain variable region comprising SEQ ID NO: 900.

[0578]

[0578] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 874 and a light chain variable region comprising SEQ ID NO: 901.

[0579]

[0579] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 875 and a light chain variable region comprising SEQ ID NO: 902

[0580]

[0580] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 876 and a light chain variable region comprising SEQ ID NO: 903.

[0581] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 877 and a light chain variable region comprising SEQ ID NO: 904.

[0581]

[0582] Tire first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 878 and a light chain variable region comprising SEQ ID NO: 905.

[0582]

[0583] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 879 and a light chain variable region comprising SEQ ID NO: 906.

[0583]

[0584] Tire first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 880 and a light chain variable region comprising SEQ ID NO: 907.

[0584]

[0585] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 881 and a light chain variable region comprising SEQ ID NO: 908.

[0585]

[0586] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 882 and a light chain variable region comprising SEQ ID NO: 909.

[0586]

[0587] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 883 and a light chain variable region comprising SEQ ID NO: 910.

[0587]

[0588] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 884 and a light chain variable region comprising SEQ ID NO: 911.

[0588]

[0589] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 885 and a light chain variable region comprising SEQ ID NO: 912.

[0589]

[0590] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 886 and a light chain variable region comprising SEQ ID NO: 913.

[0591] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 887 and a light chain variable region comprising SEQ ID NO: 914.

[0590]

[0592] Tire first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 888 and a light chain variable region comprising SEQ ID NO: 915.

[0591]

[0593] The first antigen binding domain that has specificity for an antigen on a T-cell (e.g., CD3) can comprise a heavy chain variable region comprising SEQ ID NO: 889 and a light chain variable region comprising SEQ ID NO: 916.

[0592]

[0594] Tire target antigen binding domain having specificity for EGFR and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 959, a CDR2 having the amino acid sequence of SEQ ID NO: 960, and a CDR3 having the amino acid of SEQ ID NO: 961, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 962, a CDR2 having the amino acid sequence of SEQ ID NO: 963, and a CDR3 having the amino acid of SEQ ID NO: 964. The target antigen binding domain specificity for EGFR can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 947 and a light chain variable region having the amino acid sequence of SEQ ID NO: 948.

[0593]

[0595] The target antigen binding domain having specificity for B7-H3 and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 965, a CDR2 having the amino acid sequence of SEQ ID NO: 966, and a CDR3 having tire amino acid of SEQ ID NO: 967, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 968, a CDR2 having the amino acid sequence of SEQ ID NO: 969, and a CDR3 having the amino acid of SEQ ID NO: 970. Tire target antigen binding domain having specificity B7-H3 (e.g., CD3) can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 949 and a light chain variable region having the amino acid sequence of SEQ ID NO: 950.

[0594]

[0596] The target antigen binding domain having specificity for CDH6 and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 977, a CDR2 having the amino acid sequence of SEQ ID NO: 978, and a CDR3 having the amino acid of SEQ ID NO: 979, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 980. a CDR2 having the amino acid sequence of SEQ ID NO: 981, and a CDR3 having the amino acid of SEQ ID NO: 982. The target antigen binding domain having specificity for CDH6 can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 953 and a light chain variable region having the amino acid sequence of SEQ ID NO: 954.

[0595]

[0597] Tire target antigen binding domain having specificity for EpCAM and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 983, a CDR2 having the amino acid sequence of SEQ ID NO: 984, and a CDR3 having the amino acid of SEQ ID NO: 985, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 986, a CDR2 having the amino acid sequence of SEQ ID NO: 987, and a CDR3 having the amino acid of SEQ ID NO: 988. The target antigen binding domain having specificity for EpCAM can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 955 and a light chain variable region having tire amino acid sequence of SEQ ID NO: 956.

[0596]

[0598] The target antigen binding domain having specificity for STEAP-1 and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 989, a CDR2 having the amino acid sequence of SEQ ID NO: 990, and a CDR3 having the amino acid of SEQ ID NO: 991, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 992, a CDR2 having the amino acid sequence of SEQ ID NO: 993, and a CDR3 having the amino acid of SEQ ID NO: 994. The target antigen binding domain having specificity for STEP-1 can comprise a heavy chain variable region having tire amino acid sequence of SEQ ID NO: 957 and a light chain variable region having the amino acid sequence of SEQ ID NO: 958.

[0597]

[0599] The target antigen binding domain having specificity for DLL and can comprise a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 971, a CDR2 having the amino acid sequence of SEQ ID NO: 972, and a CDR3 having the amino acid of SEQ ID NO: 973, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 974. a CDR2 having the amino acid sequence of SEQ ID NO: 975, and a CDR3 having the amino acid of SEQ ID NO: 976. Tire target antigen binding domain having specificity for DLL can comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 951 and a light chain variable region having the amino acid sequence of SEQ ID NO: 952.

[0598]

[0600] When present, the half-life extension element is preferably a sdAB that has binding specificity for human serum albumin and comprises a CDR1, a CDR2, and a CDR3. Tire CDR1 can comprise SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 37, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions. CDR2 can comprise SEQ ID NO: 2. SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 38, or a variant of any of the foregoing comprising up to about 3 am...

Claims

1. CLAIMS1. A T-cell engager comprising:(a) a first antigen binding domain that has binding specificity for an antigen on a T-cell;(b) a target antigen binding domain that has binding specificity for a target antigen;(c) a first blocking element and a second blocking element each having binding specificity for the first antigen binding domain;(d) a first protease cleavable linker that operably links the first blocking element to the first antigen binding domain and a second protease cleavable linker that operably links the second blocking element to the first antigen binding domain; and(e) an optional half-life extension element.

2. The T-cell engager of claim 1. wherein the antigen on the T-cell is CD3 epsilon. CD3 gamma. CD3 delta, CD19, CD20, CD45, CD28, CS2, CD3, CD4, LFA1, CD40, CD52, CD19, CD22, CD80, CD86, CD267, CD269, CD268, or CD32B.

3. The T-cell engager of any one of claims 1 or 2, wherein the first antigen binding domain has binding specificity to CD3 epsilon.

4. The T-cell engager of any one of the preceding claims, wherein the target antigen is an antigen associated with a cancer cell, a tumor, or a tumor-associated microenvironment.

5. The T-cell engager of any one of the preceding claims, wherein the target antigen is EGFR, PSMA, EpCAM, BCMA, 5T4, KLK2, AFP, Axl, B7-H3, Cadherin-6 (CDH6), CAIX, CD117. CD123, CD138, CD166. CD19, CD20, CD205, CD22. CD30. CD33, CD352, CD37, CD38. CD44, CD52, CD56. CD70, CD71, CD74, CD79b, CEACAM5, c-MET, DLL3, EphA2, FAP, FGFR2, FGFR3, glypican-3, FLT-3, FOLR1, gpNMB, HER2, HPV-16 E6, HPV-16 E7, ITGA3, SLC39A6, Mesothehn, Mucl, Mucl6, NaPi2b, Nectin-4, P-cadherin, Prolactin R, PSCA, PTK7, R0R1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIME Trop2, or WT1 .

6. The T-cell engager of any one of claims 1-4, wherein the target antigen is an immune modulatory protein selected from CTLA-4, CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT,TIM-1, 0X40, DNAM-1, PD-L1, PD1, PD-L2, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, VISTA, IL6-R, IL-6, TNFa, CD 19, CD20, CD22, CD52, integrin a4, integrin a4b7, CDlla, CTLA4-Ig fiision, IL-17, IL12 / 23, IL12, IL23, TGF-beta, IL-2, IL-21, IL- 18, IL2RA, IL2RB, IL2RG, IL21R, IL18R1, IL18RAP, IL12RB1, IL12RB2, CD4, 4-1BB, 4-1BBL, GITR, GITRL and a claudin, such as claudin 1, claudin 4. claudin 6, claudin 7 or claudin 18.2.

7. The T-cell engager of any one of claims 1-4, wherein the target antigen is selected from the group consisting of EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, and STEAP-1.

8. The T-cell engager of any one of tire preceding claims, wherein the first antigen binding domain and the target antigen binding domain each comprise an antibody or antigen binding fragment thereof.

9. The T-cell engager of any one of the preceding claims, wherein the first antigen binding domain is a single domain antibody, a Fab or a single-chain variable fragment (scFv).

10. The T-cell engager of any one of the preceding claims, wherein the target antigen binding domain is a single domain antibody. Fab or scFv.

11. The T-cell engager of any one of claims 1-10, wherein the first antigen binding domain is a scFv and the second antigen binding domain is a Fab.

12. The T-cell engager of any one of claims 1-10, wherein the first antigen binding domain is a Fab and the second antigen binding domain is a scFv.

13. The T-cell engager of any one of the preceding claims, wherein the first antigen binding domain and the target antigen binding domain are operably linked.

14. The T-cell engager of any one of the preceding claims, wherein the first antigen binding domain and the target antigen binding domain are operably linked through a linker.

15. The T-cell engager of claim 14, wherein the linker is a synthetic linker, a peptide linker, or a chemical bond.

16. The T-cell engager of claim 15, wherein tire linker is a peptide linker.

17. The T-cell engager of claim 16, wherein the peptide linker is a short peptide linker having about 4 amino acids to about 6 amino acids.

18. Tire T-cell engager of claim 16, wherein the peptide linker is a long peptide linker having about 5 amino acids to about 10 amino acids.

19. The T-cell engager of any one of the preceding claims, wherein the first protease cleavable linker and the second protease cleavable linker each independently comprise a sequence that is capable of being cleaved by a protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, an elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), a fibroblast activation protein (FAP), an ADAM metalloproteinase, a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease.

20. The T-cell engager of any one of the preceding claims, wherein the first protease cleavable linker and the second protease cleavable linker each independently comprise a sequence that is cleaved by cathepsin selected from the group consisting of cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, and cathepsin G.

21. The T-cell engager of any one of the preceding claims, wherein the first protease cleavable linker and the second protease cleavable linker each independently comprise a sequence that is cleaved by a matrix metalloprotease (MMP) selected from the group consisting of MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, and MMP14.

22. The T-cell engager of any one of tlie preceding claims, wherein the first protease cleavable linker and the second protease cleavable linker are cleaved by different proteases.

23. The T-cell engager of any one of claims 1-22, wherein the first protease cleavable linker and the second protease cleavable linker are cleaved by the same proteases.

24. Tire T-cell engager of any one of the preceding claims, wherein the first blocking element is a peptide from about 5 amino acids in length to about 17 amino acids in length, and the second blocking element is a peptide from about 5 amino acids in length to about 17 amino acids in length.

25. The T-cell engager of any one of claims 1-23, wherein the first blocking element is a peptide from about 7 amino acids in length to about 13 amino acids in length, and tire second blocking element is a peptide from about 7 amino acids in length to about 13 amino acids in length.

26. The T-cell engager of any one of claims 1-23, wherein the first blocking element is a peptide from about 5 amino acids in length to about 15 amino acids in length, and the second blocking element is a peptide from about 5 amino acids in length to about 15 amino acids in length.

27. The T-cell engager of any one of tire preceding claims, wherein the first blocking element and second blocking element comprise tire same amino acid sequence.

28. The T-cell engager of any of claims 1-22, wherein the first blocking element and second blocking element comprise different amino acid sequences.

29. The T-cell engager of any one of the preceding claims, wherein the first blocking element and the second blocking element each comprise an epitope for the first antigen binding domain.

30. The T-cell engager of claim 29, wherein the first blocking element and the second blocking element comprise the same or different epitopes.

31. The T-cell engager of any one of the preceding claims, wherein in the first blocking element and the second blocking element are derived from a naturally occurring polypeptide.

32. The T-cell engager of any one of the preceding claims, wherein in the first blocking element and the second blocking element are non-naturally occurring peptides.

33. Tire T-cell engager of any one of the preceding claims, wherein in the first blocking element and the second blocking element comprise non-canonical amino acids.

34. The T-cell engager of any one of the preceding claims, wherein the half-life extension element is present.

35. Tire T-cell engager of any one of the preceding claims, wherein the half-life extension element comprises a serum albumin binding domain, a serum albumin, transferrin, a transferrin binding domain, or immunoglobulin Fc, or fragment thereof36. The T-cell engager of any one of the preceding claims, wherein the half-life extension element is an antigen binding polypeptide which binds human serum albumin.

37. The T-cell engager of claim 36, wherein the antigen binding polypeptide is a single domain antibody fragment (sdAb).

38. The T-cell engager of claim 37, wherein the sdAb competes with neonatal Fc receptor (FcRn) for binding to HSA.

39. The T-cell engager of any one of claims 36-38, wherein the sdAb has binding specificity for domain one of human serum albumin.

40. The T-cell engager of any one of claims 36-39, wherein the sdAb is a heavy chain variable domain (VH), a variable heavy domain of heavy chain (VHH), a single domain shark variable domain of new antigen receptor (VNAR), or a light chain variable (VL) domain.

41. The T-cell engager further comprising a third protease cleavable linker that operably links the half-life extension element to the T-cell engager.

42. Tire T-cell engager of claim 41, wherein tire third protease cleavable linker operably links the half-life extension element to the C-terminus of the T-cell engager.

43. The T-cell engager of claim 41, wherein the third protease cleavable linker operably links the half-life extension element to the N-terminus of the T-cell engager.

44. Tire T-cell engager of claim 34, wherein the half-life extension element is an immunoglobulin Fc.

45. The T-cell engager of claim 44, wherein the immunoglobulin Fc comprises knob-in-hole heterodimerization modification.

46. Tire T-cell engager of any one of the preceding claims, further comprising a blocking element having binding specificity for the target antigen binding domain.

47. The T-cell engager of claim 46, wherein the blocking element comprises a target antigen protein or fragment thereof.

48. Tire T-cell engager of claim 46, wherein tire blocking element comprises an antigen-binding fragment selected from a single domain antibody, a Fab, or a scFv.

49. The T-cell engager of any one of the preceding claims, further comprising a second target antigen binding domain having specificity for a target antigen.

50. Tire T-cell engager of any one of claims 1-49, further comprising a third target antigen binding domain having specificity for a target antigen.

51. The T-cell engager of any one of claims 49 or 50, wherein the target antigen is a tumor antigen selected from EGFR, PSMA, EpCAM, BCMA, 5T4, KLK2, AFP, Axl, B7-H3, Cadherin-6, CAIX,CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD38, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, CEACAM5, c-MET, DLL3, EphA2, FAP, FGFR2, FGFR3, glypican-3, FLT-3, F0LR1, gpNMB, HER2, HPV-16 E6, HPV-16 E7, ITGA3, SLC39A6, Mesothelin, Mucl, Mucl6, NaPi2b, Nectin-4, P-cadherin, Prolactin R, PSCA, PTK7, R0R1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, and WT1.

52. The T-cell engager of any one claims 49 or 50, wherein the target antigen is EGFR, B7-H3, DLL-3, CDH6, PSMA, EpCAM, or STEAP-1.

53. Tire T-cell engager of any one of the preceding claims, further comprising an antigen binding domain having specificity to a T-cell co-stimulatory molecule.

54. The T-cell engager of claim 53, wherein the co-stimulatory molecule is CD28, 4-1BB, ICOS, CD27, CD2, CD40L, GITR, 0X40 and CD226.

55. The T-cell engager of any one of tire preceding claims, further comprising an antigen binding domain having specificity for an immune checkpoint protein.

56. The T-cell engager of claim 55, wherein the immune checkpoint protein is selected from the group consisting of 2B4, TIGIT, CD155, ICOS, HVEM, LIGHT, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, and VISTA.

57. The T-cell engager of any of the preceding claims, wherein the T-cell engager comprises two polypeptide chains.

51. A T-cell engager comprising(a) a first antigen binding domain comprising an antibody Fab or Fv that has binding specificity for an antigen on a T-cell;(b) a target antigen binding domain comprising an antibody Fab or Fv that has binding specificity for a target antigen;(c) a first blocking element and a second blocking element each having binding specificity for the first antigen binding domain;(d) a first protease cleavable linker that operably links the first blocking element to the first antigen binding domain and a second protease cleavable linker that operably links the second blocking element to the first antigen binding domain; and(e) an optional half-life extension element; wherein the T cell engager comprises:(i) a first polypeptide comprising:(i) a first heavy chain Fab fragment (VH + CHI) or a first heavy chain variable domain (VH) ;(ii) a second heavy chain Fab fragment (VH + CHI) or a second heavy chain variable domain (VH);(iii) a first blocking element having specificity for the first antigen binding domain; and(iv) a first protease cleavable linker that operably links the first blocking element to the first antigen binding domain or target antigen binding domain;(b) a second polypeptide comprising:(i) a first light chain Fab fragment (VL + CL) or light chain variable domain (VL), that is complementary to the first heavy chain Fab fragment (VH + CHI) or a first heavy chain variable domain (VH) of the first polypeptide;(ii) the second light chain Fab fragment (VL + CL), or light chain variable domain (VL), that is complementary to second heavy chain Fab fragment (VH + CHI) or a second heavy chain variable domain (VH) of the second polypeptide;(iii) a second blocking element having specificity for the first antigen binding domain;(iv) a second protease cleavable linker that operably links the second blocking element to the first antigen binding domain.

52. Tire T-cell engager of claim 51, further comprising a half-life extension element.

53. The T-cell engager of claim 52, wherein the half-life extension element is serum albumin or a serum albumin peptide or an scFv or sdAb that binds serum albumin.

54. The T-cell engager of claim 53, wherein the half-life extension element is an scFv or sdAb that binds serum albumin and the first polypeptide and / or the second polypeptide comprise the scFv or sdAb.55 Tire T-cell engager of claim 52, wherein tire first polypeptide further comprises the scFv or sdAb.

56. The T-cell engager of claim 55, wherein the first polypeptide further comprises an Fc region and the scFv or sdAb is linked to the Fc region.

57. Tire T-cell engager of claim 52, wherein the first polypeptide comprises a first heavy chain antibody Fab fragment (VH + CHI) and a second heavy chain variable domain (VH).

58. The T-cell engager of claim 52, wherein the second polypeptide comprises a first light chain antibody Fab fragment (VL + CL) that is complimentary to the VH + CHI on the first polypeptide and a second VH that is complimentary to the VL on the second polypeptide.

59. The T-cell engager of claim 52, wherein the first polypeptide comprises a first heavy chain variable domain (VH) and a second heavy chain antibody Fab fragment (VH + CHI).

60. The T-cell engager of claim 52, wherein the second polypeptide comprises a first light chain variable domain (VL) that is complimentary to the first VH on the first polypeptide and a second light chain antibody Fab fragment (VL + CL) that is complimentary to the second VH + CHI.

61. Tire T-cell engager of claim 52, wherein the antigen on the T-cell is CD3 epsilon, CD3 gamma, CD3 delta, CD19. CD20, CD45. CD28, CS2, CD3, CD4, LFAL CD40, CD52, CD19. CD22, CD80, CD86, CD267, CD269, CD268, or CD32B61. The T-cell engager of claim 52, wherein the first antigen binding domain has binding specificity to CD3 epsilon.

62. The T-cell engager of any one claims 52, wherein the target antigen is an antigen associated with a cancer cell, a tumor, or a tumor-associated microenvironment.

63. The T-cell engager of any one claims 52-62, wherein the target antigen is EGFR, PSMA, EpCAM, BCMA, 5T4, KLK2, AFP, Axl, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD38, CD44, CD52, CD56, CD70, CD71, CD74. CD79b, CEACAM5, c-MET, DLL3, EphA2. FAP, FGFR2, FGFR3, glypican-3, FLT-3, FOLR1, gpNMB, HER2. HPV-16 E6, HPV-16 E7, ITGA3. SLC39A6, Mesothelin. Mucl, Mucl6, NaPi2b. Nectin- 4, P-cadherm, Prolactin R, PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, orWTl .

64. Tire T-cell engager of any one claims 52-62, wherein the target antigen is selected from the group consisting of EGFR. B7-H3, DLL-3, PSMA, CDH6, EpCAM. and STEAP-1.

65. The T-cell engager of any one of any one claims 52-64, wherein the first antigen binding domain and the target antigen binding domain are operably linked.

66. The T-cell engager of claim 65, wherein the linker is a synthetic linker, a peptide linker, or a chemical bond.

67. The T-cell engager of claim 65, wherein the linker is a peptide linker.

68. Tire T-cell engager of claim 67, wherein the peptide linker is a short peptide linker having about 4 amino acids to about 6 amino acids.

69. The T-cell engager of claim 67, wherein the peptide linker is a long peptide linker having about 5 amino acids to about 10 amino acids.

70. Tire T-cell engager of any one claims 52-69, wherein the first protease cleavable linker and the second protease cleavable linker each independently comprise a sequence that is capable of being cleaved by a protease selected from the group consisting of a kallikrein, thrombin, chymase. carboxypeptidase A, an elastase, PR-3, granzyme M, a calpain. a matrix metalloproteinase (MMP). a fibroblast activationprotein (FAP), an ADAM metalloproteinase, a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease.

71. Tire T-cell engager of any one of claims 52-71, wherein the first blocking element is a peptide from about 5 amino acids in length to about 17 amino acids in length, and the second blocking element is a peptide from about 5 amino acids in length to about 17 amino acids in length.

72. The T-cell engager of any one of claims 52-71, wherein the first blocking element is a peptide from about 7 amino acids in length to about 13 amino acids in length, and the second blocking element is a peptide from about 7 amino acids in length to about 13 amino acids in length.

73. The T-cell engager of any one of claims 52-71, wherein the first blocking element is a peptide from about 5 amino acids in length to about 15 amino acids in length, and the second blocking element is a peptide from about 5 amino acids in length to about 15 amino acids in length.

75. The T-cell engager of claim 52, wherein the first polypeptide further comprises a blocking element having specificity for the target antigen binding domain.

76. The T-cell engager of claim 75, wherein the blocking element is operably linked to the VH + CHI or VH through a linker.

77. The T-cell engager of any one of claims 75 or 76, wherein the blocking element comprises a target antigen protein or fragment thereof.

78. The T-cell engager of any one of claims 75 or 76, wherein the blocking element comprises an antigen-binding fragment selected from a single domain antibody, a Fab, or a scFv.

79. The T-cell engager of claim 52, wherein the first polypeptide further comprises one or more additional target antigen binding domains having specificity for a target antigen.

80. The T-cell engager of claim 52, wherein the one or more additional target antigen binding domains are scFv’s.

81. Tire T-cell engager of claim 52, wherein the second polypeptide further comprises one or more additional target antigen binding domains having specificity for a target antigen.

82. The T-cell engager of claim 52, wherein the one or more additional target antigen binding domains are scFv’s.

83. Tire T-cell engager of any one of claims 79-82, wherein the target antigen is EGFR, B7-H3, DLL- 3, CDH6, PSMA, EpCAM, or STEAP-1.

84. The T-cell engager of any one of the preceding claims, wherein the T-cell engager comprises a structure shown in any one of FIGs. 3A-3F, 17A-17P, and 18A-18L, 19A-19I, 20A-20I, 29A-29C, and 39A-34B.

85. The T-cell engager of any one of tire preceding claims, wherein the first antigen binding domain comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 999, a CDR2 having the amino acid sequence of SEQ ID NO: 1000, and a CDR3 having the amino acid of SEQ ID NO: 1001, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1002, a CDR2 having the amino acid sequence of SEQ ID NO: 1003, and a CDR3 having the amino acid of SEQ ID NO: 1004.

86. The T-cell engager of any one of claims 52-85, wherein the first antigen binding domain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 995 and a light chain variable region having tire amino acid sequence of SEQ ID NO: 996.

87. The T-cell engager of any one of claims 52-84, wherein the first antigen binding domain comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1005. a CDR2 having the amino acid sequence of SEQ ID NO: 1006, and a CDR3 having the amino acid of SEQ ID NO: 1007, and a light chain variable region comprising a CDR1 having the amino acidsequence of SEQ ID NO: 1008, a CDR2 having the amino acid sequence of SEQ ID NO: 1009, and a CDR3 having the amino acid of SEQ ID NO: 1010.

88. The T-cell engager of any one of the preceding claims, wherein the first antigen binding domain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 997 and a light chain variable region having the amino acid sequence of SEQ ID NO: 998.

89. The T-cell engager of any one of the preceding claims, wherein the target antigen binding domain comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 959, a CDR2 having the amino acid sequence of SEQ ID NO: 960, and a CDR3 having the amino acid of SEQ ID NO: 961, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:

962. a CDR2 having the amino acid sequence of SEQ ID NO: 963, and a CDR3 having the amino acid of SEQ ID NO: 964.

90. Tire T-cell engager of any one of the preceding claims, wherein the target antigen binding domain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 947 and a light chain variable region having the amino acid sequence of SEQ ID NO: 948.

91. The T-cell engager of any one of claims 52-90, wherein the target antigen binding domain comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 965, a CDR2 having the amino acid sequence of SEQ ID NO: 966, and a CDR3 having the amino acid of SEQ ID NO: 967, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:

968. a CDR2 having the amino acid sequence of SEQ ID NO: 969, and a CDR3 having the amino acid of SEQ ID NO: 970.

92. The T-cell engager of any one of claims 52-90, wherein the target antigen binding domain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 949 and a light chain variable region having the amino acid sequence of SEQ ID NO: 950.

93. The T-cell engager of any one of claims 52-90, wherein the target antigen binding domain comprises a heavy chain variable region comprising a CDR1 having tire amino acid sequence of SEQ ID NO: 977, a CDR2 having the amino acid sequence of SEQ ID NO: 978, and a CDR3 having the amino acid of SEQ ID NO: 979, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:

980. a CDR2 having tire amino acid sequence of SEQ ID NO: 981, and a CDR3 having the amino acid of SEQ ID NO: 982.

94. The T-cell engager of any one of claims 52-90, wherein the target antigen binding domain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 953 and a light chain variable region having the amino acid sequence of SEQ ID NO: 954.

95. The T-cell engager of any one of claims 52-90, wherein the target antigen binding domain comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 983, a CDR2 having the amino acid sequence of SEQ ID NO: 984, and a CDR3 having the amino acid of SEQ ID NO: 985, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 986, a CDR2 having the amino acid sequence of SEQ ID NO: 987, and a CDR3 having the amino acid of SEQ ID NO: 988.

96. The T-cell engager of any one of claims 52-90, wherein the target antigen binding domain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 955 and a light chain variable region having tire amino acid sequence of SEQ ID NO: 956.

97. The T-cell engager of any one of claims 52-90, wherein the target antigen binding domain comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 989, a CDR2 having the amino acid sequence of SEQ ID NO: 990, and a CDR3 having the amino acid of SEQ ID NO: 991, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 992, a CDR2 having the amino acid sequence of SEQ ID NO: 993, and a CDR3 having the amino acid of SEQ ID NO: 994.

98. The T-cell engager of any one of claims 52-90, wherein the target antigen binding domain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 957 and a light chain variable region having the amino acid sequence of SEQ ID NO: 958.

99. The T-cell engager of any one of claims 52-90, wherein the target antigen binding domain comprises a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 971, a CDR2 having the amino acid sequence of SEQ ID NO: 972, and a CDR3 having the amino acid of SEQ ID NO: 973, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 974, a CDR2 having the amino acid sequence of SEQ ID NO: 975, and a CDR3 having the amino acid of SEQ ID NO: 976.

100. The T-cell engager of any one of claims 52-90, wherein the target antigen binding domain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 951 and a light chain variable region having tire amino acid sequence of SEQ ID NO: 952.

101. The T-cell engager of any one of the preceding claims, wherein the first heavy chain variable region and the second heavy chain variable region are operably linked w ith a peptide comprising an ammo acid sequence of SEQ ID NO:

920. SEQ ID NO: 921, SEQ ID NO: 923, SEQ ID NO: 924.

102. The T-cell engager of any one of the preceding claims, wherein the first light chain variable region and the first heavy chain variable region are operably linked with a peptide comprising an amino acid sequence of SEQ ID NO: 920, SEQ ID NO: 924, SEQ ID NO: 926, or SEQ ID NO: 927.

103. The T-cell engager of any one of tire preceding claims, wherein the first blocking element and second blocking element comprise the amino acid sequence of SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 496, and SEQ ID NO: 497, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 496, and SEQ ID NO: 497.

104. The T-cell engager of any one of the preceding claims, wherein the protease cleavable linker comprises the amino acid sequence selected from SEQ ID NOs: 510 to 535, 707, 708, 942, 943, 944, 945, 946, or 1011.

105. The T-cell engager of any one of the preceding claims, wherein the half-life extension element is a sdAb that has binding specificity for human serum albumin comprising a CDR1, CDR2 and CDR3; wherein CDR1 comprises any one of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 37, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; CDR2 comprises any one of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 38, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; and CDR3 comprises any one of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 12 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions106. The T-cell engager of claim 105, wherein the sdAb comprises:(1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to about 3 amino acid substitutions;(2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 amino acid substitutions;(3) a CDR1 comprising the amino acid sequence of SEQ ID NO:

7. or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;(4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;(5) a CDR1 comprising the amino acid sequence of SEQ ID NO:

7. or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;(6) a CDR1 comprising the amino acid sequence of SEQ ID NO:

7. or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;(7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO:

9. or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;(8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising tire amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

12. or a variant thereof comprising up to about 3 amino acid substitutions;(9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;(10) a CDRl comprising the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;(11) a CDR1 comprising the amino acid sequence of SEQ ID NO:

8. or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;(12) a CDR1 comprising the amino acid sequence of SEQ ID NO:

8. or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions;(13) a CDR1 comprising the amino acid sequence of SEQ ID NO:

8. or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions; or(14) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising tire amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

12. or a variant thereof comprising up to about 3 amino acid substitutions.

107. The T-cell engager of any one of tire preceding claims, wherein the wherein the T-cell engager comprises:(1) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 716; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 824;(2) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 716; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 825;(3) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 717; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 826;(4) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 717; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 825;(5) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 719; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(6) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 721; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(7) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 723; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(8) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 725; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 466;(9) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 727; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 828;(10) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 729; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 830;(11) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 731; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 832;(12) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 734; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 835;(13) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 734; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 836;(14) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 735; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 835;(15) first poly peptide chain that comprises the amino acid sequence of SEQ ID NO: 735; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 836;(16) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 737; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(17) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 739; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(18) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 741; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(19) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 743; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(20) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 748; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 837;(21) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 750; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 837;(22) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 753; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(23) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 755; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(24) a first poly peptide chain that comprises the amino acid sequence of SEQ ID NO: 757; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(25) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 759; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(26) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 764; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 841;(27) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 764; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 842;(28) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 766; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 841;(29) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 775; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 845;(30) a first poly peptide chain that comprises the amino acid sequence of SEQ ID NO: 775; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 846;(31) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 777; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 845;(32) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 777; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 846;(33) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 777; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464;(34) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 779; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464;(35) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 782; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(36) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 784; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 697;(37) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 788; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 849;(38) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 788; ; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 850;(39) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 792; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 849;(40) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 792; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 850;(59) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 799; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 852;(60) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 801; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 854;(61) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 803; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 856;(62) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 809; and) a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 859;(63) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 809; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 860;(64) first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 811; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 859;(65) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 811; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 860;(66) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 815; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 861;(67) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 816; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 861;(68) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 819; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 861;(69) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 820; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 861;(70) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 821; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 464; or(71) a first polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 822; and a second polypeptide chain that comprises the amino acid sequence of SEQ ID NO: 862; or a variant of any of tire foregoing.

108. A T-cell engager comprising:(a) a first antigen binding domain that has binding specificity for CD3 comprising a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 999, a CDR2 having the amino acid sequence of SEQ ID NO: 1000, and a CDR3 having the amino acid of SEQ ID NO: 1001 and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1002, a CDR2 having the amino acid sequence of SEQ ID NO: 1003, and a CDR3 having the amino acid of SEQ ID NO: 1004 or a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1005, a CDR2 having the amino acid sequence of SEQ ID NO: 1006. and a CDR3 having the amino acid of SEQ ID NO: 1007, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1008, a CDR2 having the amino acid sequence of SEQ ID NO: 1009, and a CDR3 having the amino acid of SEQ ID NO: 1010;(b) a target antigen binding domain comprising a heavy chain variable region comprising a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 959, a CDR2 having the amino acid sequence of SEQ ID NO: 960, and a CDR3 having the amino acid of SEQ ID NO: 961, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ IDNO: 962, a CDR2 having the amino acid sequence of SEQ ID NO: 963, and a CDR3 having the amino acid of SEQ ID NO: 964; a heavy chain variable region comprising a CDR1 having tire amino acid sequence of SEQ ID NO: 965, a CDR2 having the amino acid sequence of SEQ ID NO: 966, and a CDR3 having the amino acid of SEQ ID NO: 967, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:

968. a CDR2 having tire amino acid sequence of SEQ ID NO: 969, and a CDR3 having the amino acid of SEQ ID NO: 970; a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 977, a CDR2 having the amino acid sequence of SEQ ID NO: 978, and a CDR3 having the amino acid of SEQ ID NO: 979, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO:

980. a CDR2 having tire amino acid sequence of SEQ ID NO: 981, and a CDR3 having the amino acid of SEQ ID NO: 982; a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 983, a CDR2 having the amino acid sequence of SEQ ID NO: 984, and a CDR3 having the amino acid of SEQ ID NO: 985, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 986, a CDR2 having the amino acid sequence of SEQ ID NO: 987, and a CDR3 having the amino acid of SEQ ID NO: 988; or a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 971, a CDR2 having the amino acid sequence of SEQ ID NO: 972, and a CDR3 having the amino acid of SEQ ID NO: 973, and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 974, a CDR2 having the amino acid sequence of SEQ ID NO: 975, and a CDR3 having the amino acid of SEQ ID NO: 976;(c) a first blocking element and a second blocking element each having binding specificity for tire first antigen binding domain; wherein the first and second blocking element comprise the amino acid sequence of SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 496, or SEQ ID NO: 497.(d) a first protease cleavable linker that operably links the first blocking element to the first antigen binding domain and a second protease cleavable linker that operably links the second blocking element to the first antigen binding domain, wherein the protease cleavable linker comprises an amino acid sequence selected from SEQ ID NOs: 510 to 535, 707. 708, 942. 943, 944. 945, 946. or 1011; and(e) an optional half-life extension element; wherein the half-life extension element comprises a CDR1, CDR2 and CDR3; wherein CDR1 comprises any one of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 37, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; CDR2 comprises any one of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 38, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; and CDR3 comprises any one of SEQ ID NO:

3. SEQ ID NO:

6. SEQ ID NO: 12 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions.

109. Tire T-cell engager of claim 108, wherein the first antigen binding domain and the target antigen binding domain, each independently comprise VH and VL or VH + CHI and VL + CL.

110. The T-cell engager of any one of claims 108-109, wherein the first antigen binding domain and the target domain can be operably linked through a linker having an amino acid sequence of SEQ ID NO: 920, SEQ ID NO: 921, SEQ ID NO: 923, SEQ ID NO: 924 SEQ ID NO: 926, or SEQ ID NO: 927, and combinations thereof.

111. The T-cell engager of any one of claims 108-109, wherein the VH or VH + CHI of the first antigen binding domain and the VH or VH + CHI of the target antigen binding domain are operably linked through a linker having the amino acid sequence of SEQ ID NO: 920, SEQ ID NO: 921, SEQ ID NO: 923, or SEQ ID NO: 924.

112. The T-cell engager of any one of claims 108-109, wherein the VL or VL + CL of the first antigen binding domain and the VL + CL of the target antigen binding domain are operably linked through a linker having the amino acid sequence of SEQ ID NO: 920, SEQ ID NO:

924. SEQ ID NO: 926, or SEQ ID NO: 927.

113. Tire T cell engager of claim 108, wherein the T cell engager comprises a first polypeptide and a second polypeptide,(a) the first polypeptide comprising(i) a first heavy chain Fab fragment (VH + CHI);(ii) a target antigen binding domain comprising an scFV that bind target antigen; and(iii) a first blocking element having specificity for the first antigen binding domain: and(iv) a first protease cleavable linker that operably links the first blocking element to the first antigen binding domain or the scFV that binds target antigen;(b) the second polypeptide comprising:(i) a first light chain Fab fragment (VL + CL)(iii) a second blocking element having specificity for the first antigen binding domain; and(iv) a second protease cleavable linker that operably links the second blocking element to the first antigen binding domain.

114. Tire T-cell engager of claim 113, further comprising a half life extension element.

115. The T-cell engager of claim 114, wherein the half life extension element is serum albumin or a serum albumin peptide or an scFv or dAb that binds serum albumin.

116. Tire T-cell engager of claim 114, wherein the half life extension element is an scFv or dAb that binds serum albumin and tire first polypeptide and / or tire second polypeptide comprise the scFv or dAb that binds serum albumin.

117. The T-cell engager of claim 113, wherein the first polypeptide further comprises the scFv or dAb.

118. Tire T-cell engager of claim 117, wherein the scFv or dAb is operably linked to CHI of the first heavy chain Fab fragment or operably linked to the scFV that binds target antigen.

119. The T-cell engager of claim 117, wherein the scFv or scFv or dAb is operably linked to CHI of the first heavy chain Fab fragment or operably linked to the scFV that binds target antigen through a linker that is optionally protease clevable.

120. The T-cell engager of any one of tire preceding claims, wherein the T-cell engager comprises a structure shown in any one of FIGs. 17J or 17L.

121. A nucleic acid encoding the T-cell engager of any one of the preceding claims.

122. The nucleic acid of claim 121, wherein the nucleic acid is RNA.

123. Tire nucleic acid of claims 121 or 122, wherein RNA is mRNA or circular RNA (circRNA).

124. A vector comprising the nucleic acid of any one of claims 121-123.

125. The vector of claim 124, wherein the vector is selected from the group consisting of adeno- associated virus (AAV), retroviral vector, lentiviral vector, and adenoviral vector.

126. A host cell comprising the nucleic acid of any one of claims 121-123, or tire vector of claims 124 or 125.

127. A pharmaceutical composition comprising the T-cell engager of any one of claims 1-120, the nucleic acid of any one of claims 121-123, the vector of claims 124 or 125, or the host cell of claim 126.

128. A method of treating a cancer, comprising administering to a subject in need thereof an effective amount of the T-cell engager of any one of claims 1-120, the nucleic acid of any one of claims 121-123. the vector of claims 124 or 125, or the host cell of claim 126.