T cell engager antibody-drug conjugates

Abstract

Provided herein are, inter alia, protein complexes having the functionality of a T cell engager (TCE) as well as an antibody-drug conjugate (ADC). These protein complexes provided surprisingly effective mechanism of action for cancer treatment as compared to traditional TCEs (e.g., bispecific antibodies) as they exhibit high specificity and effectivity while lacking undesirable adverse side effects.

Description

[0001] Attorney Docket No. 60846-0002W01

[0002] T CELL ENGAGER ANTIBODY-DRUG CONJUGATES

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This disclosure claims priority to and benefit of U.S. Provisional Patent Application Serial No. 63 / 708,148, filed October 16, 2024, which is incorporated herein by reference in its entirety.

[0005] TECHNICAL FIELD

[0006] This disclosure relates to protein complexes for cancer treatment, and methods of use thereof.

[0007] BACKGROUND

[0008] T cell engagers (TCEs) are a type of immunotherapy designed to enhance the body's immune system's ability to fight cancer. They are usually bispecific antibodies that bind to both a tumor cell antigen and a T cell receptor, bringing the two cells into close proximity and activating the T cell to kill the cancer cell. Antibody-drug conjugate (ADC) is typically composed of a monoclonal antibody covalently attached to a cytotoxic drug via a chemical linker. It combines both the advantages of highly specific targeting ability and highly potent killing effect to achieve accurate and efficient elimination of cancer cells, which has become one of the hotspots for the research and development of anticancer drugs.

[0009] There is a need in the art for cancer therapeutics that harness the whole immune system to target cancer. The recombinant polypeptides and methods of use provided herein address this and other needs in the art.

[0010] SUMMARY

[0011] The disclosure provides protein complexes that can form one or more T cell engagers (TCEs) and / or one or more antibody-drug conjugates (ADCs) upon cleavage, e.g., by a protease within tumor microenvironment (TME), thereby significantly improving cancer treatment efficacy by harnessing the benefits of these two types of molecules. Before reaching TME, the TCE function of the protein complexes can be effectively masked, thereby further improving the safety profile. To achieve these goals, the protein complexes disclosed herein have multiple structural features. For example, one or more cancer antigen-binding moieties provide tight binding with improved selectivity; detuned effector cell antigen (e.g., Attorney Docket No. 60846-0002W01

[0012] CD3) binding allows a moderate TCE function to achieve a balance of efficacy and safety; optimized substrate sequences of the protease provide both effective masking of the TCE function before cleavage and similar cleavage kinetics as compared to a clinical benchmark; stably linked drugs minimize free payload toxicity; and silenced Fc region avoids nonspecific uptake and toxicity.

[0013] Experiments of the present disclosure demonstrate that the protein complexes exhibited a synergistic effect to kill target cancer cells through both the TCE function and ADC function when combined with effector cells (e.g., PBMCs). The effective masking of the TCE function before protease-induced cleavage was also demonstrated, which is critical to minimize undesirable T cell activation. Further, no effector cell killing by the protein complexes was observed, indicating that the protein complexes have a greater safety profile as compared to traditional TCE molecules.

[0014] Also provided herein are antibodies, antigen-binding fragments thereof, protein complexes thereof, and ADCs thereof that bind to DLL3 or CD3.

[0015] In one aspect, the disclosure is related to a protein complex comprising: (a) a fragment crystallizable (Fc) region; (b) a first antigen-binding moiety that specifically binds to a first cancer antigen; and (c) a second antigen-binding moiety that specifically binds to an effector cell antigen, In some embodiments, the second antigen-binding moiety is linked to the Fc region via a first linker peptide. In some embodiments, the first antigen-binding moiety is linked to the second antigen-binding moiety. In some embodiments, at least one therapeutic agent is conjugated to the Fc region, optionally the at least one therapeutic agent is a cytotoxic or cytostatic agent. In some embodiments, the first linker peptide is cleavable by a protease within tumor microenvironment. In some embodiments, the first antigenbinding moiety and the second antigen-binding moiety form a T cell engager (TCE) when the first linker peptide is cleaved. In some embodiments, the protein complex described herein can function as an antibody-drug conjugate before cleavage. In some embodiments, the first antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or multispecific antibody (e.g., a bispecific antibody). In some embodiments, the first cancer antigen is CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD96, CD123, CDH17, CEA, CLDN6, CLDN18.2, cMET, CCR4, DLL3, DLL4, DKK, NECTIN4, Claudin, LIVE6, BCMA, HER2, HER3, HLA-G, KLK2, LGR5, MAGEA4 / 8, mesothelin, angiopoietin, FGF, FGFR, MUC16, PRAME, PSMA, STEAP1, TMEFF2, PD-L1, EGFR, EGFRvIII, ENPP3, EpCAM, gplOO, GPC3, GUCY2C, VEGF, VEGFR2, CTLA-4, PD-1, RANK-L, GD2, SLAMF7, PDGFRA, TROP-2, FR-alpha, FcRH5, FLT3, GPRC5D, ROR1, Attorney Docket No. 60846-0002W01

[0016] 5T4, B7-H3, or B7-H4. In some embodiments, the second antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or a multi-specific antibody (e.g., a bispecific antibody). In some embodiments, the effector cell antigen is CD3, CD28, PD-1, TIGIT, Lag3, CD16A, CD16B, CD32A, CD32B, CD32C, CD64, or SIRPa. In some embodiments, the Fc region comprises: (a) a first polypeptide comprising an optional first hinge region, a first CH2 domain, and a first CH3 domain, and (b) a second polypeptide comprising an optional second hinge region, a second CH2 domain, and a second CH3 domain, in some embodiments, the first and second polypeptides associate with each other, forming the Fc region.

[0017] In some embodiments, the protein complex described herein further comprises: a third antigen-binding moiety that specifically binds to a second cancer antigen, and a fourth antigen-binding moiety that specifically binds to the effector cell antigen, in some embodiments, the fourth antigen-binding moiety is linked to the Fc region via a second linker peptide. In some embodiments, the third antigen-binding moiety is linked to the fourth antigen-binding moiety. In some embodiments, the second linker peptide is cleavable by a protease within tumor microenvironment. In some embodiments, the third antigen-binding moiety and the fourth antigen-binding moiety form a T cell engager (TCE) when the second linker peptide is cleaved. In some embodiments, the third antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or multi-specific antibody (e.g., a bispecific antibody). In some embodiments, the second cancer antigen is CD 19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD96, CD123, CDH17, CEA, CLDN6, CLDN18.2, cMET, CCR4, DLL3, DLL4, DKK, NECTIN4, Claudin, LIVE6, BCMA, HER2, HER3, HLA-G, KLK2, LGR5, MAGEA4 / 8, mesothelin, angiopoietin, FGF, FGFR, MUC16, PRAME, PSMA, STEAP1, TMEFF2, PD-L1, EGFR, EGFRvIII, ENPP3, EpCAM, gplOO, GPC3, GUCY2C, VEGF, VEGFR2, CTLA-4, PD-1, RANK-L, GD2, SLAMF7, PDGFRA, TROP-2, FR-alpha, FcRH5, FLT3, GPRC5D, ROR1, 5T4, B7-H3, or B7-H4. In some embodiments, the fourth antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or a multi-specific antibody (e.g., a bispecific antibody). In some embodiments, the first cancer antigen and the second cancer antigen are the same. In some embodiments, the first and third antigen-binding moieties are a VHH, and / or in some embodiments, the second and fourth antigen-binding moieties are an scFv.

[0018] In some embodiments, the protein complex described herein further comprises: a fifth antigen-binding moiety that specifically binds to a third cancer antigen, and a sixth antigenbinding moiety that specifically binds to a fourth cancer antigen; optionally the fifth antigenbinding moiety is linked to the first antigen-binding moiety, and the sixth antigen-binding Attorney Docket No. 60846-0002W01 moiety is linked to the third antigen-binding moiety. In some embodiments, the fifth and / or sixth antigen-binding moi eties are an scFv, a Fab, a single domain antibody (e.g., a VHH), or multi-specific antibody (e.g., a bispecific antibody). In some embodiments, the first, third, fifth and sixth antigen-binding moieties are a VHH, and / or in some embodiments, the second and fourth antigen-binding moieties are an scFv.

[0019] In some embodiments, the protein complex described herein further comprises: a third antigen-binding moiety that specifically binds to a second cancer antigen; in some embodiments, the third antigen-binding moiety is linked to the Fc region. In some embodiments, the third antigen-binding moiety and the Fc region form an antibody-drug conjugate (ADC) when the first linker peptide is cleaved. In some embodiments, the third antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or multispecific antibody (e.g., a bispecific antibody). In some embodiments, the third cancer antigen is CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD96, CD123, CDH17, CEA, CLDN6, CLDN18.2, cMET, CCR4, DLL3, DLL4, DKK, NECTIN4, Claudin, LIVE6, BCMA, HER2, HER3, HLA-G, KLK2, LGR5, MAGEA4 / 8, mesothelin, angiopoietin, FGF, FGFR, MUC16, PRAME, PSMA, STEAP1, TMEFF2, PD-L1, EGFR, EGFRvIII, ENPP3, EpCAM, gplOO, GPC3, GUCY2C, VEGF, VEGFR2, CTLA-4, PD-1, RANK-L, GD2, SLAMF7, PDGFRA, TROP-2, FR-alpha , FcRH5, FLT3, GPRC5D, ROR1, 5T4, B7-H3, or B7-H4. In some embodiments, the first cancer antigen and the second cancer antigen are the same. In some embodiments, the first antigen-binding moiety is a VHH, and / or in some embodiments, the second and third antigen-binding moieties are an scFv.

[0020] In some embodiments, the protein complex described herein further comprises: a fourth antigen-binding moiety that specifically binds to a third cancer antigen; in some embodiments, the fourth antigen-binding moiety is linked to the first antigen-binding moiety. In some embodiments, the fourth antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or multi-specific antibody (e.g., a bispecific antibody). In some embodiments, the first and fourth antigen-binding moieties are a VHH, and / or in some embodiments, the second and third antigen-binding moieties are an scFv.

[0021] In some embodiments, the protein complex described herein further comprises: a fourth antigen-binding moiety that specifically binds to a third cancer antigen; in some embodiments, the fourth antigen-binding moiety is linked to the third antigen-binding moiety. In some embodiments, the fourth antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or multi-specific antibody (e.g., a bispecific antibody). In some Attorney Docket No. 60846-0002W01 embodiments, the first and second antigen-binding moieties are an scFv, and / or in some embodiments, the third and fourth antigen-binding moieties are a VHH.

[0022] In some embodiments, the effector cell antigen is CD3. In some embodiments, the Fc region does not induce antibody effector functions, e.g., antibody-dependent cellular cytotoxicity (ADCC).

[0023] In one aspect, the disclosure is related to a protein complex comprising: (a) a fragment crystallizable (Fc) region, in some embodiments, at least one therapeutic agent is conjugated to the Fc region; (b) a first antigen-binding moiety that specifically binds to a first cancer antigen and a second antigen-binding moiety that specifically binds to an effector cell antigen, in some embodiments, the first antigen-binding moiety is linked to the second antigen-binding moiety, in some embodiments, the second antigen-binding moiety is linked to the Fc region via a first linker peptide; and (c) a third antigen-binding moiety that specifically binds to a second cancer antigen and a fourth antigen-binding moiety that specifically binds to the effector cell antigen, in some embodiments, the third antigen-binding moiety is linked to the fourth antigen-binding moiety, in some embodiments, the fourth antigen-binding moiety is linked to the Fc region via a second linker peptide; in some embodiments, the first antigen-binding moiety and the second antigen-binding moiety form a first T cell engager (TCE) when the first linker peptide is cleaved, and in some embodiments, the third antigen-binding moiety and the fourth antigen-binding moiety form a second TCE when the second linker peptide is cleaved.

[0024] In one aspect, the disclosure is related to a protein complex comprising: (a) a fragment crystallizable (Fc) region, in some embodiments, at least one therapeutic agent is conjugated to the Fc region; (b) a first antigen-binding moiety that specifically binds to a first cancer antigen and a second antigen-binding moiety that specifically binds to an effector cell antigen, in some embodiments, the first antigen-binding moiety is linked to the second antigen-binding moiety, in some embodiments, the second antigen-binding moiety is linked to the Fc region via a first linker peptide; and (c) a third antigen-binding moiety that specifically binds to a second cancer antigen, in some embodiments, the third antigen-binding moiety is linked to the Fc; in some embodiments, when the first linker peptide is cleaved, the first antigen-binding moiety and the second antigen-binding moiety form a T cell engager (TCE), and the third antigen-binding and the Fc region form an antibody-drug conjugate (ADC). In some embodiments, the protein complex forms an ADC before the first and / or second linker peptides are cleaved. Attorney Docket No. 60846-0002W01

[0025] In one aspect, the disclosure is related to a protein complex comprising: (a) a first polypeptide comprising, optionally form N-terminus to C-terminus: (1) a first antigenbinding moiety that specifically binds to a first cancer antigen, (2) a second antigen-binding moiety that specifically binds to a first effector cell antigen, (3) a first linker peptide, (4) an optional first hinge region, (5) a first CH2 domain, and (6) a first CH3 domain; (b) a second polypeptide comprising, optionally form N-terminus to C-terminus: (1) a third antigenbinding moiety that specifically binds to a second cancer antigen, (2) a fourth antigen-binding moiety that specifically binds to a second effector cell antigen, (3) a second linker peptide, (4) an optional second hinge region, (5) a second CH2 domain, and (6) a second CH3 domain; in some embodiments, the first and second linker peptides are cleavable by a protease within tumor microenvironment. In some embodiments, the protein complex described herein further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) therapeutic agent conjugated to the first and / or second polypeptides. In some embodiments, the first antigenbinding moiety and the second antigen-binding moiety form a first T cell engager (TCE) when the first linker peptide is cleaved, and in some embodiments, the third antigen-binding moiety and the fourth antigen-binding moiety form a second T cell engager (TCE) when the second linker peptide is cleaved.

[0026] In one aspect, the disclosure is related to a protein complex comprising: (a) a first polypeptide comprising, optionally form N-terminus to C-terminus: (1) a first antigenbinding moiety that specifically binds to a first cancer antigen, (2) a second antigen-binding moiety that specifically binds to a second cancer antigen, (3) a third antigen-binding moiety that specifically binds to a first effector cell antigen, (4) a first linker peptide, (5) an optional first hinge region, (6) a first CH2 domain, and (7) a first CH3 domain; and (b) a second polypeptide comprising, optionally form N-terminus to C-terminus: (1) a fourth antigenbinding moiety that specifically binds to a third cancer antigen, (2) a fifth antigen-binding moiety that specifically binds to a fourth cancer antigen, (3) a sixth antigen-binding moiety that specifically binds to a second effector cell antigen, (4) a second linker peptide, (5) an optional second hinge region, (6) a second CH2 domain, and (7) a second CH3 domain; in some embodiments, the first and second linker peptides are cleavable by a protease within tumor microenvironment. In some embodiments, the protein complex described herein further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) therapeutic agent conjugated to the first and / or second polypeptides. In some embodiments, the first, second, and third antigenbinding moieties form a first T cell engager (TCE) when the first linker peptide is cleaved, Attorney Docket No. 60846-0002W01 and in some embodiments, the fourth, fifth, and sixth antigen-binding moieties form a second T cell engager (TCE) when the second linker peptide is cleaved.

[0027] In one aspect, the disclosure is related to a protein complex comprising: (a) a first polypeptide comprising, optionally form N-terminus to C-terminus: (1) a first antigenbinding moiety that specifically binds to a first cancer antigen, (2) a second antigen-binding moiety that specifically binds to an effector cell antigen, (3) a first linker peptide, (4) an optional first hinge region, (5) a first CH2 domain, and (6) a first CH3 domain; and (b) a second polypeptide comprising, optionally form N-terminus to C-terminus: (1) a third antigen-binding moiety that specifically binds to a second cancer antigen, (2) an optional second hinge region, (3) a second CH2 domain, and (4) a second CH3 domain; in some embodiments, the first linker peptide is cleavable by a protease within tumor microenvironment. In some embodiments, the protein complex further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) therapeutic agent conjugated to the first and / or second polypeptides. In some embodiments, when the first linker peptide is cleaved, the first antigenbinding moiety and the second antigen-binding moiety form a T cell engager (TCE) and the third antigen-binding moiety, the optional first hinge region, the optional second hinge region, the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain together form an antibody-drug conjugate (ADC).

[0028] In one aspect, the disclosure is related to a protein complex comprising: (a) a first polypeptide comprising, optionally form N-terminus to C-terminus: (1) a first antigenbinding moiety that specifically binds to a first cancer antigen, (2) a second antigen-binding moiety that specifically binds to a second cancer antigen, (3) a third antigen-binding moiety that specifically binds to an effector cell antigen, (4) a first linker peptide, (5) an optional first hinge region, (6) a first CH2 domain, and (7) a first CH3 domain; and (b) a second polypeptide comprising, optionally form N-terminus to C-terminus: (1) a fourth antigenbinding moiety that specifically binds to a third cancer antigen, (2) an optional second hinge region, (3) a second CH2 domain, and (4) a second CH3 domain; in some embodiments, the first linker peptide is cleavable by a protease within tumor microenvironment. In some embodiments, the protein complex described herein further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) therapeutic agent conjugated to the first and / or second polypeptides. In some embodiments, when the first linker peptide is cleaved, the first, second, and third antigen-binding moieties form a T cell engager (TCE) and the fourth antigen-binding moiety, the optional first hinge region, the optional second hinge region, the first CH2 domain, the Attorney Docket No. 60846-0002W01 second CH2 domain, the first CH3 domain, and the second CH3 domain together form an antibody-drug conjugate (ADC).

[0029] In one aspect, the disclosure is related to a protein complex comprising: (a) a first polypeptide comprising, optionally form N-terminus to C-terminus: (1) a first antigenbinding moiety that specifically binds to a first cancer antigen, (2) a second antigen-binding moiety that specifically binds to an effector cell antigen, (3) a first linker peptide, (4) an optional first hinge region, (5) a first CH2 domain, and (6) a first CH3 domain; and (b) a second polypeptide comprising, optionally form N-terminus to C-terminus: (1) a third antigen-binding moiety that specifically binds to a second cancer antigen, (2) a fourth antigen-binding moiety that specifically binds to a third cancer antigen, (3) an optional second hinge region, (4) a second CH2 domain, and (5) a second CH3 domain; in some embodiments, the first linker peptide is cleavable by a protease within tumor microenvironment. In some embodiments, the protein complex described herein further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) therapeutic agent conjugated to the first and / or second polypeptides. In some embodiments, when the first linker peptide is cleaved, the first and second antigen-binding moieties form a T cell engager (TCE), and the third antigen-binding moiety, the fourth antigen-binding moiety, the optional first hinge region, the optional second hinge region, the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain together form an antibody-drug conjugate (ADC).

[0030] In one aspect, the disclosure is related to a nucleic acid comprising a polynucleotide encoding the protein complex described herein. In some embodiments, the nucleic acid is a DNA (e.g., cDNA) or RNA (e.g., mRNA).

[0031] In one aspect, the disclosure is related to a vector comprising one or more of the nucleic acids described herein.

[0032] In one aspect, the disclosure is related to a cell comprising the vector described herein. In some embodiments, the cell is a CHO cell. In one aspect, the disclosure is related to a cell comprising the nucleic acid described herein.

[0033] In one aspect, the disclosure is related to a method of producing a protein complex, the method comprising (1) culturing the cell described herein under conditions sufficient for the cell to produce the protein complex; and (2) collecting the protein complex produced by the cell.

[0034] In one aspect, the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex described herein, to the subject. In some embodiments, the Attorney Docket No. 60846-0002W01 subject has a cancer cell expressing a cancer antigen (e.g., DLL3). In some embodiments, the cancer is breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, diffuse large B-cell lymphoma, mesothelioma, lung cancer, ovarian cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, cholangiocarcinoma, head and neck cancer, blood cancer, or a combination thereof.

[0035] In one aspect, the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the protein complex described herein. In one aspect, the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the protein complex described herein.

[0036] In one aspect, the disclosure is related to a pharmaceutical composition comprising the protein complex described herein, and a pharmaceutically acceptable carrier.

[0037] Attorney Docket No.: 60846-0002W01

[0038] In one aspect, the disclosure is related to a recombinant polypeptide comprising: (i) a cancer antigen binding domain bound to an effector cell binding domain; (ii) an Fc dimerizing domain covalently bound to at least one therapeutic moiety; and (iii) a protease cleavable linker connecting said cancer antigen binding domain with said Fc dimerizing domain through binding said effector cell binding domain to said Fc dimerizing domain. In some embodiments said cancer antigen binding domain is bound to said effector cell binding domain through a chemical linker.

[0039] In some embodiments, said cancer antigen binding domain is an antibody domain. In some embodiments, said cancer antigen binding domain is an scFv, a Fab, a single domain antibody or a bispecific antibody. In some embodiments, said single domain antibody is a VHH. In some embodiments, said single domain antibody is a llama VHH, a camelid VHH, or a shark VHH. In some embodiments, said cancer antigen is an adult acute lymphoblastic leukemia (ALL) antigen, chronic lymphocytic leukemia (CLL) antigen, non-Hodgkin lymphoma (NHL) antigen, adult acute myeloid leukemia (AML) antigen, multiple myeloma (MML) antigen, leukemia antigen, lymphoma antigen, urothelial carcinoma antigen, colorectal cancer antigen, non-small cell lung carcinoma (NSCLC) antigen, lung cancer antigen, glioblastoma antigen, gastric cancer antigen, hepatocellular carcinoma antigen, metastatic melanoma antigen, endometrial cancer antigen, secondary bone cancer antigen, neuroblastoma antigen, soft tissue carcinoma antigen, breast cancer antigen, cervical cancer antigen, bladder cancer antigen, nasopharyngeal cancer antigen, or ovarian cancer antigen. In some embodiments, said cancer antigen is CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD96, CD123, CDH17, CEA, CLDN6, CLDN18.2, cMET, CCR4DLL3, DLL4, DKK, NECTIN4, Claudin, LIVE6, BCMA, HER2, HER3, HLA-G, KLK2, LGR5, MAGEA4 / 8, mesothelin, angiopoietin, FGF, FGFR, MUC16, PRAME, PSMA, STEAP1, TMEFF2, PD-L1, EGFR, EGFRvIII, ENPP3, EpCAM, gplOO, GPC3, GUCY2C, VEGF, VEGFR2, CTLA-4, PD-1, RANK-L, GD2, SLAMF7, PDGFRA, TROP-2, FR-alpha , FcRH5, FLT3, GPRC5D, ROR1, 5T4, B7-H3, or B7-H4. In some embodiments, said effector cell binding domain is a CD3 binding domain, a CD28 binding domain, a PD-1 binding domain, a TIGIT binding domain, a Lag3 binding domain, a CD16A binding domain, a CD16B binding domain, a CD32A binding domain, a CD32B binding domain, a CD32C binding domain, a CD64 binding domain, or a SIRP-alpha binding domain. In some embodiments, said effector cell binding domain is a CD3 binding domain. In some Attorney Docket No.: 60846-0002W01 embodiments, said effector cell binding domain is a scFv or a VHH. In some embodiments, said effector cell binding domain comprises the sequence of SEQ ID NO: 1.

[0040] In some embodiments, said Fc dimerizing domain comprises a CH2 domain and a CH3 domain. In some embodiments, said Fc dimerizing domain does not induce antibodydependent cellular cytotoxicity (ADCC) relative to a standard control. In some embodiments, said Fc dimerizing domain comprises a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, a cysteine at a position corresponding to position 302, an alanine at a position corresponding to position 234, or an alanine at a position corresponding to position 235. In some embodiments, said Fc dimerizing domain comprises the sequence of SEQ ID NO: 2.

[0041] In some embodiments, said protease cleavable linker has a length of about 2 to about 500 amino acids. In some embodiments, said protease cleavable linker has a length of about 5 to about 20 amino acids. In some embodiments, said protease is a serum protease or a tumor microenvironment protease. In some embodiments, said protease is a matrix metalloprotease, an ADAM protease, a caspase protease, a PSA protease, a calpain protease, a legumain protease, a Fap protease, a DPPIV protease, or a PEP protease. In some embodiments, said tumor microenvironment protease is MMP-2, MMP-9 or MMP-14. In some embodiments, said tumor microenvironment protease has the sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, said serum protease is a thrombin protease, a plasmin protease, a trypsin protease, a chymotrypsin protease, a factor Xa protease, a factor Xlla protease, a factor Xia protease, a factor IXa protease, a kallikrein protease, a complement Cis protease, a complement Clr protease, a cathepsin protease, an elastase, a carboxypeptidase, an angiotensin-converting enzyme (ACE), a granzyme protease, or a MASP1 protease. In some embodiments, said protease cleavable linker is a serum protease cleavable linker. In some embodiments, said protease cleavable linker is a tumor microenvironment protease cleavable linker. In some embodiments, said tumor microenvironment protease cleavable linker is a matrix metalloprotease (MMP) cleavable linker.

[0042] In some embodiments, said at least one therapeutic moiety is a small molecule, a peptide, or a nucleic acid. In some embodiments, said at least one therapeutic moiety is a microtubule targeting therapeutic moiety, a DNA damaging therapeutic moiety, an RNA polymerase inhibiting therapeutic moiety, or a topoisomerase inhibiting therapeutic moiety. Attorney Docket No.: 60846-0002W01

[0043] In some embodiments, said at least one therapeutic moiety is a plurality of therapeutic moieties.

[0044] In some embodiments, said polypeptide is a single chain polypeptide.

[0045] In some embodiments, said cancer antigen binding domain is a DLL3 binding domain and said effector cell binding domain is a CD3 binding domain. In some embodiments, said cancer antigen binding domain is a NECTIN-4 binding domain and said effector cell binding domain is a CD3 binding domain.

[0046] In some embodiments, said at least one therapeutic moiety is Dxd, TM, or MMAE.

[0047] In some embodiments, said polypeptide is a first single chain polypeptide. In some embodiments, said cancer antigen binding domain is a first cancer antigen binding domain, said effector cell binding domain is a first effector cell binding domain, said Fc dimerizing domain is a first Fc dimerizing domain, said plurality of therapeutic moieties is a first plurality of therapeutic moieties and said protease cleavable linker is a first protease cleavable linker.

[0048] In some embodiments, said first Fc dimerizing domain is bound to a second recombinant polypeptide comprising: (i) a second cancer antigen binding domain bound to a second effector cell binding domain through a second chemical linker; (ii) a second Fc dimerizing domain covalently bound to at least one second therapeutic moiety; and (iii) a second protease cleavable linker connecting said second cancer antigen binding domain with said second Fc dimerizing domain through binding said second effector cell binding domain to said second Fc dimerizing domain; in some embodiments, said first Fc dimerizing domain and said second Fc dimerizing domain are covalently bound together thereby binding said first recombinant polypeptide to said second recombinant polypeptide.

[0049] In some embodiments, said second cancer antigen binding domain is an antibody domain. In some embodiments, said second effector cell binding domain binding domain is an antibody domain. In some embodiments, said second cancer antigen binding domain is an scFv, a Fab, a single domain antibody or a bispecific antibody. In some embodiments, said single domain antibody is a VHH. In some embodiments, said single domain antibody is a llama VHH, a camelid VHH, or a shark VHH. In some embodiments, said second cancer antigen is an adult acute lymphoblastic leukemia (ALL) antigen, chronic lymphocytic leukemia (CLL) antigen, non-Hodgkin lymphoma (NHL) antigen, adult acute myeloid leukemia (AML) antigen, multiple myeloma (MML) antigen, leukemia antigen, lymphoma Attorney Docket No.: 60846-0002W01 antigen, urothelial carcinoma antigen, colorectal cancer antigen, non-small cell lung carcinoma (NSCLC) antigen, lung cancer antigen, glioblastoma antigen, gastric cancer antigen, hepatocellular carcinoma antigen, metastatic melanoma antigen, endometrial cancer antigen, secondary bone cancer antigen, neuroblastoma antigen, soft tissue carcinoma antigen, breast cancer antigen, cervical cancer antigen, bladder cancer antigen, nasopharyngeal cancer antigen, or ovarian cancer antigen. In some embodiments, said second cancer antigen is CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD96, CD123, CDH17, CEA, CLDN6, CLDN18.2, cMET, CCR4DLL3, DLL4, DKK, NECTIN4, Claudin, LIVE6, BCMA, HER2, HER3, HLA-G, KLK2, LGR5, MAGEA4 / 8, mesothelin, angiopoietin, FGF, FGFR, MUC16, PRAME, PSMA, STEAP1, TMEFF2, PD-L1, EGFR, EGFRvIII, ENPP3, EpCAM, gplOO, GPC3, GUCY2C, VEGF, VEGFR2, CTLA-4, PD-1, RANK-L, GD2, SLAMF7, PDGFRA, TROP-2, FR-alpha , FcRH5, FLT3, GPRC5D, ROR1, 5T4, B7-H3, or B7-H4. In some embodiments, said second effector cell binding domain is a CD3 binding domain, a CD28 binding domain, a PD-1 binding domain, a TIGIT binding domain, a Lag3 binding domain, a CD16A binding domain, a CD16B binding domain, a CD32A binding domain, a CD32B binding domain, a CD32C binding domain, a CD64 binding domain, or a SIRP-alpha binding domain. In some embodiments, said second effector cell binding domain is a CD3 binding domain. In some embodiments, said effector cell binding domain is a scFv or a VHH. In some embodiments, said second effector cell binding domain comprises the sequence of SEQ ID NO: 1.

[0050] In some embodiments, said second Fc dimerizing domain comprises a CH2 domain and a CH3 domain. In some embodiments, said second Fc dimerizing domain does not induce antibody-dependent cellular cytotoxicity (ADCC) relative to a standard control. In some embodiments, said second Fc dimerizing domain comprises a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, a cysteine at a position corresponding to position 302, an alanine at a position corresponding to position 234, or an alanine at a position corresponding to position 235. In some embodiments, said second Fc dimerizing domain comprises the sequence of SEQ ID NO: 2.

[0051] In some embodiments, said second protease cleavable linker has a length of about 2 to about 500 amino acids. In some embodiments, said second protease cleavable linker has a length of about 5 to about 20 amino acids. In some embodiments, said second protease is a serum protease or a tumor microenvironment protease. In some embodiments, said second Attorney Docket No.: 60846-0002W01 protease is a matrix metalloprotease, an ADAM protease, a caspase protease, a PSA protease, a calpain protease, a legumain protease, a Fap protease, a DPPIV protease, or a PEP protease. In some embodiments, said tumor microenvironment protease is MMP-2, MMP-9 or MMP- 14. In some embodiments, said tumor microenvironment protease has the sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, said serum protease is a thrombin protease, a plasmin protease, a trypsin protease, a chymotrypsin protease, a factor Xa protease, a factor Xlla protease, a factor Xia protease, a factor IXa protease, a kallikrein protease, a complement Cis protease, a complement Clr protease, a cathepsin protease, an elastase, a carboxypeptidase, an angiotensin-converting enzyme (ACE), a granzyme protease, or a MASP1 protease. In some embodiments, said second protease cleavable linker is a serum protease cleavable linker. In some embodiments, said second protease cleavable linker is a tumor microenvironment protease cleavable linker. In some embodiments, said tumor microenvironment protease cleavable linker is a matrix metalloprotease (MMP) cleavable linker.

[0052] In some embodiments, said second therapeutic moiety is a small molecule, a peptide, or a nucleic acid. In some embodiments, said second therapeutic moiety is a microtubule targeting therapeutic moiety, a DNA damaging therapeutic moiety, an RNA polymerase inhibiting therapeutic moiety, or a topoisomerase inhibiting therapeutic moiety. In some embodiments, said at least one second therapeutic moiety is a plurality of second therapeutic moieties.

[0053] In some embodiments, said second polypeptide is a single chain polypeptide.

[0054] In some embodiments, said second cancer antigen binding domain is a DLL3 binding domain and said second effector cell binding domain is a CD3 binding domain. In some embodiments, said second cancer antigen binding domain is a NECTIN-4 binding domain and said second effector cell binding domain is a CD3 binding domain.

[0055] In some embodiments, said at least one second therapeutic moiety is Dxd, TM, or MMAE.

[0056] In some embodiments, said first Fc dimerizing domain comprises a first CH2 domain and a first CH3 domain. In some embodiments, said second Fc dimerizing domain comprises a second CH2 domain and a second CH3 domain. In some embodiments, said first CH2 domain is covalently bound to said second CH2 domain. In some embodiments, said first CH2 domain is covalently bound to said second CH2 domain through a disulfide linkage. In Attorney Docket No.: 60846-0002W01 some embodiments, said first Fc dimerizing domain and said second Fc dimerizing domain form an antibody Fc region.

[0057] In some embodiments, said recombinant polypeptide forms part of a T cell.

[0058] In one aspect, the disclosure is related to an isolated nucleic acid encoding the recombinant polypeptide described herein.

[0059] In one aspect, the disclosure is related to an expression vector comprising the nucleic acid described herein. In some embodiments, said expression vector is a viral vector. In some embodiments, said virus is a lentivirus or onco-retrovirus.

[0060] In one aspect, the disclosure is related to a T lymphocyte comprising the expression vector described herein.

[0061] In one aspect, the disclosure is related to method of treating cancer in a subject in need thereof, said method comprising administering to a subject a therapeutically effective amount of a recombinant polypeptide described herein, thereby treating cancer in said subject.

[0062] In one aspect, the disclosure is related to a pharmaceutical composition comprising a therapeutically effective amount of a recombinant polypeptide described herein and a pharmaceutically acceptable excipient.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0064] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0065] BRIEF DESCRIPTION OF DRAWINGS

[0066] FIG. 1 shows a schematic structure of an exemplary TCE-ADC molecule described herein. Attorney Docket No.: 60846-0002W01

[0067] FIG. 2 shows schematic diagrams of the mechanism of action and benefits of the exemplary TCE-ADC molecules described herein. In the symmetric cleavage diagram, Structure A (“A”) has ADC function but no TCE function due to masked anti-CD3 moieties; Structure B (“B”) has anti-cancer / anti-CD3 moieties exposed post-MMP substrate cleavage and has TCE function; and Structure C (“C”) lacks target-specific binding and clears quickly. In the asymmetric cleavage diagram, Structure A (“A”) has ADC function but no TCE function due to a masked anti-CD3 moiety; Structure B (“B”) has anti-cancer / anti-CD3 moieties exposed post-MMP substrate cleavage and has TCE function; and Structure C (“C”) acts as a monovalent ADC post-MMP substrate cleavage.

[0068] FIG. 3 is a table showing BLI epitope binning assay results.

[0069] FIG. 4 is a table showing BLI kinetic affinity assay results. Benchmark molecules AMG-757 analog and INN analog were used as controls. Human or monkey DLL3 was used as the antigen.

[0070] FIG. 5 shows the binding percentage of different anti-DLL3 binders binding to DLL3- expressing SHP-77 cells. Benchmark molecules AMG-757 analog and INN analog were used as controls.

[0071] FIGS. 6A-6B show the binding percentage and MFI of different anti-DLL3 binders binding to DLL3 -expressing SHP-77 cells. Benchmark molecules AMG-757 analog, INN analog, and an isotype control were used as controls.

[0072] FIG. 7 shows the predicted complex of MMP2 substrate pep5 and the catalytic domain of MMP2. The hydrophobic interaction between Phe5 from MMP2 active site and Leu2 from pep5 (PLGLAG; SEQ ID NO: 8) is shows as a dashed line.

[0073] FIGS. 8A-8E show SDS-PAGE gel images of MMP2-cleavable proteins being cleaved by MMP2 at different enzyme concentrations.

[0074] FIGS. 9A-9B show schematic structures of T cell engager-antibody-drug conjugates (TCE-ADC) molecules targeting one or more cancer antigens and CD3. The anti-CD3 moieties are represented by black boxes, and anti-cancer moieties are represented by empty boxes. Cleavable linkers are represented by circles and MMP cleavage sites are indicated by a dashed line. Drugs conjugated at the Fc region are represented by stars. Each of the anticancer moieties can be an scFv, a Fab, or a VHH. Each of the anti-CD3 moieties can be an scFv, a Fab, or a VHH. Attorney Docket No.: 60846-0002W01

[0075] FIGS. 10 A- 10C show the binding percentage of P1-P5 binding to 293 T, 293T-DLL3, and Jurkat cells, respectively. Benchmark molecule AMG-757 analog was used as a control.

[0076] FIG. 11 is a table showing the T cell activation strength of masked (uncleaved) and unmasked (cleaved) P2 and P3. Schematic structures of the masked and unmasked molecules are provided.

[0077] FIG. 12 shows the percentage of living SHP-77 cells incubated with P2 or P2- Exatecan, either with or without PBMCs. Different concentrations of P2 (without conjugated payload) or P2-Exatecan were used during incubation. An isotype control was used as a negative control.

[0078] FIG. 13 shows the percentage of living CD2-positive cells incubated with different P2-ADCs in the presence of PBMCs. Different concentrations of P2 (without conjugated payload) or P2-Exatecan were used during incubation. An isotype control was used as a negative control.

[0079] FIG. 14 shows schematic representation of exemplary TCE-ADC molecules provided herein including embodiments thereof including VHH, scFv and Fab combinations.

[0080] FIG. 15 shows schematic representation of the mechanism of action of the TCE-ADC molecules provided herein including embodiments thereof.

[0081] FIG. 16 lists CDR sequences of VHHs under Kabat definition described in the disclosure. VVH1-VHH16 are anti-DLL3 VHHs. VHH17-VHH30 are anti-CD3 VHHs.

[0082] FIG. 17 lists CDR sequences of VHHs under Chothia definition described in the disclosure. VVH1-VHH16 are anti-DLL3 VHHs. VHH17-VHH30 are anti-CD3 VHHs.

[0083] FIG. 18 lists amino acid sequences of VHHs described in the disclosure. VVH1- VHH16 are anti-DLL3 VHHs. VHH17-VHH30 are anti-CD3 VHHs.

[0084] FIG. 19 lists VH and VL CDR sequences of scFvs under Kabat definition described in the disclosure. ScFvl-ScFvl6 are anti-CD3 scFvs. ScFvl7-ScFv22 are anti-DLL3 scFvs.

[0085] FIG. 20 lists VH and VL CDR sequences of scFvs under Chothia definition described in the disclosure. ScFvl-ScFvl6 are anti-CD3 scFvs. ScFvl7-ScFv22 are anti-DLL3 scFvs.

[0086] FIG. 21 lists amino acid sequences of scFvs described in the disclosure and the corresponding VH and VL. ScFvl-ScFvl6 are anti-CD3 scFvs. ScFvl7-ScFv22 are anti-

[0087] DLL3 scFvs.

[0088] FIG. 22 lists additional sequences discussed in the disclosure. Attorney Docket No.: 60846-0002W01

[0089] DETAILED DESCRIPTION

[0090] The T cell engager antibody-drug conjugates (TCE-ADC molecules) provided herein including embodiments thereof provide for a safer anti -cancer targeting, from the get-go ADC activity leading to cancer killing and initiates potent immune responses. The tumor microenvironment leads to proteolytic cleavage of the TCE from the ADC and the ADC and T cell activation acts synergistically at cancer cell killing. The provided T cell engager antibody-drug conjugates exhibit increased safety (detuned anti-CD3 that is structurally rigid until MMP cleave, which represents a new method of masking). Furthermore, the nanobodies’ size allows for better tumor penetration (about 1 / 3 of Janux’s TRACTr size). The multiple mechanisms of action of the recombinant polypeptides provided herein provide for effective cancer cell killing.

[0091] Compared to traditional TCEs and ADCs the recombinant polypeptides provided herein have a synergistic mechanism of action including two functional features: (i) T cells are drawn into the tumor microenvironment in response to the release of new cancer antigens following ADC-mediated cancer cell destruction; and (ii) TCE recruits and activates these new local T cells, continuing cancer cell elimination and facilitates the penetration of ADC through the solid tumor, layer by layer. The recombinant polypeptides provided herein including embodiments thereof improve safety and efficacy and lack undesired side effects, as the TCE-ADC has no CD3 binding function before it reaches the tumor microenvironment (TME). Furthermore, the IgG like modality enables IgG like half-life followed by short postcleavage half-life. Exemplary features of the TCE-ADC molecules described herein are summarized in FIG. 1, and schematic representation of the mechanism of action of the TCE- ADC molecules are provided in FIG. 15.

[0092] As shown in FIG. 2, two approaches may be used to design the TCE-ADC molecules. Under a symmetric cleavage approach, the masked TCE-ADC molecule has ADC function but no TCE function due to masked anti-CD3 moieties. Following MMP substrate cleavage, one fragment is turned into an activated TCE and the other fragment can be cleared quickly as a side product. Under an asymmetric approach, the masked TCE-ADC molecule has ADC function but not TCE function due to a masked anti-CD3 moiety. Following MMP substrate cleavage, one fragment is turned into an activated TCE and the other fragment acts as a monoclonal ADC. Attorney Docket No.: 60846-0002W01

[0093] DEFINITIONS

[0094] As used herein, the term "about" means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term "about" means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about means the specified value.

[0095] A “chemical linker,” as provided herein, is a covalent linker, a non-covalent linker, a peptide or peptidyl linker (a linker including a peptide moiety), a cleavable peptide linker, a substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene or any combination thereof.

[0096] The chemical linker as provided herein may be a bond, -O-, -S-, -C(O)-, -C(O)O-, - C(O)NH-, -S(O)2NH-, -NH-, -NHC(O)NH-, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted alkylene, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted heteroalkylene, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted cycloalkylene, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted heterocycloalkylene, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted arylene or substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted heteroarylene.

[0097] The chemical linker as provided herein may be a bond, -O-, -S-, -C(O)-, -C(O)O- , -C(O)NH-, -S(O)2NH-, -NH-, -NHC(O)NH-, substituted or unsubstituted (e.g., C1-C20, Ci- C10, C1-C5) alkylene, substituted or unsubstituted (e.g., 2 to 20 membered, 2 to 10 membered, 2 to 5 membered) heteroalkylene, substituted or unsubstituted (e.g., Cs-Cs, C3-C6, C3-C5) cycloalkylene, substituted or unsubstituted (e.g., 3 to 8 membered, 3 to 6 membered, 3 to 5 membered) heterocycloalkylene, substituted or unsubstituted (e.g., Ce-Cio, Ce-Cs, Ce-Cs) arylene or substituted or unsubstituted (e.g., 5 to 10 membered, 5 to 8 membered, 5 to 6 membered,) heteroarylene. Attorney Docket No.: 60846-0002W01

[0098] In embodiments, the chemical linker is a covalent linker. In embodiments, the chemical linker is a hydrocarbon linker. In embodiments, the chemical linker is a cleavable peptide linker.

[0099] Thus, a chemical linker as provided herein may include a plurality of chemical moieties, wherein each of the plurality of chemical moieties is chemically different. Alternatively, the chemical linker may be a non-covalent linker. Examples of non-covalent linkers include without limitation, ionic bonds, hydrogen bonds, halogen bonds, van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), and hydrophobic interactions. In embodiments, a chemical linker is formed using conjugate chemistry including, but not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).

[0100] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0101] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions Attorney Docket No.: 60846-0002W01 thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and mini circle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0102] Nucleic acids, including e.g., nucleic acids with a phosphothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amio acid on a protein or polypeptide through a covalent, non-covalent or other interaction.

[0103] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press) as well as modifications to the nucleotide bases such as in 5 -methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos. 5,235,033 Attorney Docket No.: 60846-0002W01 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0104] Nucleic acids can include nonspecific sequences. As used herein, the term "nonspecific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0105] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.

[0106] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego. Attorney Docket No.: 60846-0002W01

[0107] A "labeled protein or polypeptide" is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the labeled protein or polypeptide may be detected by detecting the presence of the label bound to the labeled protein or polypeptide. Alternatively, methods using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin.

[0108] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, / .< ., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0109] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0110] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid po lymers and non-naturally occurring amino acid polymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety. Attorney Docket No.: 60846-0002W01

[0111] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that may be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N- terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0112] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue. For example, a selected residue in a selected antibody (or Fab domain) corresponds to light chain threonine at Kabat position 40, when the selected residue occupies the same essential spatial or other structural relationship as a light chain threonine at Kabat position 40. In some embodiments, where a selected protein is aligned for maximum homology with the light chain of an antibody (or Fab domain), the position in the aligned selected protein aligning with threonine 40 is said to correspond to threonine 40. Instead of a primary sequence alignment, a three dimensional structural alignment can also be used, e.g., where the structure of the selected protein is aligned for maximum correspondence with the light chain threonine at Kabat position 40, and the overall structures compared. In this case, an amino acid that occupies the same essential position as threonine 40 in the structural model is said to correspond to the threonine 40 residue.

[0113] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid Attorney Docket No.: 60846-0002W01 sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0114] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0115] The following eight groups each contain amino acids that are conservative substitutions for one another:

[0116] 1) Alanine (A), Glycine (G);

[0117] 2) Aspartic acid (D), Glutamic acid (E);

[0118] 3) Asparagine (N), Glutamine (Q);

[0119] 4) Arginine (R), Lysine (K);

[0120] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0121] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0122] 7) Serine (S), Threonine (T); and

[0123] 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). Attorney Docket No.: 60846-0002W01

[0124] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., of the entire polypeptide sequences of the invention or individual domains of the polypeptides of the invention), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.

[0125] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0126] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0127] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same Attorney Docket No.: 60846-0002W01 number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat ’I. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0128] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Awe. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positivevalued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults Attorney Docket No.: 60846-0002W01 a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0129] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873- 5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0130] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0131] "CD3" as referred to herein includes any of the recombinant or naturally-occurring forms of the Cluster of Differentiation 3 (CD3) proteins or variants or homologs thereof that comprise the CD3 complex that mediates signal transduction and maintains CD3 complex activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the CD3 complex). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3 proteins in the CD3 complex. In embodiments, the CD3 protein is substantially identical to the protein identified by the UniProt reference number P04234 or a variant or homolog having substantial identity thereto. In embodiments, the CD3 protein is substantially identical to the protein identified by the UniProt reference number P09693 or a variant or homolog having substantial identity thereto. In embodiments, Attorney Docket No.: 60846-0002W01 the CD3 protein is substantially identical to the protein identified by the UniProt reference number P07766 or a variant or homolog having substantial identity thereto.

[0132] "CD 16" as referred to herein includes any of the recombinant or naturally-occurring forms of the Cluster of Differentiation 16 (CD 16) protein, also known as low affinity immunoglobulin gamma Fc region receptor III-A, or variants or homologs thereof that maintain CD 16 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD 16). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD 16 protein. In embodiments, the CD 16 protein is substantially identical to the protein identified by the UniProt reference number P08637 or a variant or homolog having substantial identity thereto.

[0133] A "CD32 protein" as referred to herein includes any of the recombinant or naturally- occurring forms of the Cluster of Differentiation 32 (CD32) protein, also known as low affinity immunoglobulin gamma Fc region receptor II- A, or variants or homologs thereof that maintain CD32 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD32). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD32 protein. In embodiments, the CD32 protein is substantially identical to the protein identified by the UniProt reference number P12318 or a variant or homolog having substantial identity thereto.

[0134] A "NKp46 protein" as referred to herein includes any of the recombinant or naturally- occurring forms of the NKp46 protein, also known as natural cytotoxicity triggering receptor 1, or variants or homologs thereof that maintain NKp46 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to NKp46). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring NKp46 protein. In embodiments, the NKp46 protein is substantially identical to the protein identified by the UniProt reference number 076036 or a variant or homolog having substantial identity thereto. Attorney Docket No.: 60846-0002W01

[0135] "CD 19" as referred to herein includes any of the recombinant or naturally-occurring forms of the Cluster of Differentiation 19 (CD 19) protein, also known as B-lymphocyte antigen CD19, B-Lymphocyte Surface Antigen B4, T-Cell Surface Antigen Leu-12, CVID3 or variants or homologs thereof that maintain CD 19 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD 19). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD 19 protein. In embodiments, the CD 19 protein is substantially identical to the protein identified by the UniProt reference number P15397 or a variant or homolog having substantial identity thereto. In embodiments, the CD 19 protein is substantially identical to the protein identified by the UniProt reference number P25918 or a variant or homolog having substantial identity thereto.

[0136] "CD20" as referred to herein includes any of the recombinant or naturally-occurring forms of CD20, also known as B-lymphocyte antigen CD20, or variants or homologs thereof that maintain CD20 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD20). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD20 protein. In embodiments, the CD20 protein is substantially identical to the protein identified by the UniProt reference number Pl 1836 or a variant or homolog having substantial identity thereto.

[0137] "Her2" as referred to herein includes any of the recombinant or naturally-occurring forms of the human epidermal growth factor receptor 2 protein, also known as receptor tyrosine-protein kinase erbB-2, or variants or homologs thereof that maintain Her2 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Her2). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Her2 protein. In embodiments, the Her2 protein is substantially identical to the protein identified by the UniProt reference number P04626 or a variant or homolog having substantial identity thereto. Attorney Docket No.: 60846-0002W01

[0138] "CD123" as referred to herein includes any of the recombinant or naturally-occurring forms of CD123, also known as interleukin 3 receptor, alpha, or variants or homologs thereof that maintain CD123 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD123). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD 123 protein. In embodiments, the CD 123 protein is substantially identical to the protein identified by the UniProt reference number P26951 or a variant or homolog having substantial identity thereto.

[0139] "Tumor-associated glycoprotein 72" (TAG72) as referred to herein includes any of the recombinant or naturally-occurring forms of TAG72, or variants or homologs thereof that maintain TAG72 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to TAG72). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring TAG72 protein.

[0140] "Carcinoembryonic antigen" (CEA) as referred to herein describes a set of highly related glycoproteins involved in cell adhesion and includes any of the recombinant or naturally-occurring forms of CEA variants or homologs thereof that maintain CEA activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CEA). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to naturally occurring CEA proteins. In embodiments, the CEA protein is substantially identical to the protein identified by the UniProt reference number QI 3984 or a variant or homolog having substantial identity thereto.

[0141] The term "isolated", when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid Attorney Docket No.: 60846-0002W01 chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0142] Antibodies are large, complex molecules (molecular weight of -150,000 or about 1320 amino acids) with intricate internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region, involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system. The light and heavy chain variable regions (also referred to herein as light chain variable (VL) domain and heavy chain variable (VH) domain, respectively) come together in 3 -dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions (“CDRs”). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3- dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework ("FR"), which forms the environment for the CDRs.

[0143] The term "antibody" is used according to its commonly known meaning in the art. Antibodies exist, e.g., as intact immunoglobulins or as a number of well -characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CHI by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those Attorney Docket No.: 60846-0002W01 identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0144] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL), variable light chain (VL) domain or light chain variable region and variable heavy chain (VH), variable heavy chain (VH) domain or heavy chain variable region refer to these light and heavy chain regions, respectively. The terms variable light chain (VL), variable light chain (VL) domain and light chain variable region as referred to herein may be used interchangeably. The terms variable heavy chain (VH), variable heavy chain (VH) domain and heavy chain variable region as referred to herein may be used interchangeably. The Fc (i.e. fragment crystallizable region) is the "base" or "tail" of an immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. In embodiments, the Fc region includes a constant heavy chain domain 3 (CH3 domain) and a constant heavy chain domain 2 (CH2 domain).

[0145] The epitope of an antibody is the region of its antigen to which the antibody binds. Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a lx, 5x, lOx, 20x or lOOx excess of one antibody inhibits binding of the other by at least 30% but preferably 50%, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50: 1495, 1990). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0146] The term "antigen" as provided herein refers to molecules capable of binding to the antibody binding domain provided herein. An "antigen binding domain" as provided herein is Attorney Docket No.: 60846-0002W01 a region of an antibody that binds to an antigen (epitope). As described above, the antigen binding domain is generally composed of one constant and one variable domain of each of the heavy and the light chain (VL, VH, CL and CHI, respectively). The paratope or antigenbinding site is formed on the N-terminus of the antigen binding domain. The two variable domains of an antigen binding domain typically bind the epitope on an antigen.

[0147] For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)). "Monoclonal" antibodies (mAb) refer to antibodies derived from a single clone. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0148] For preparation of suitable antibodies of the invention and for use according to the invention, e.g., recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Patent 4,946,778, U.S. Patent No. 4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; Attorney Docket No.: 60846-0002W01

[0149] 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93 / 08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121 :210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Patent No. 4,676,980 , WO 91 / 00360; WO 92 / 200373; and EP 03089).

[0150] Methods for humanizing or primatizing non-human antibodies are well known in the art (e.g., U.S. Patent Nos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87 / 02671; EP Patent Application 0173494; Jones et al. (1986) Nature 321 :522; and Verhoyen et al. (1988) Science 239: 1534). Humanized antibodies are further described in, e.g., Winter and Milstein (1991) Nature 349:293. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Morrison et al., PNAS USA, 81 :6851-6855 (1984), Jones et al., Nature 321 :522- 525 (1986); Riechmann et al., Nature 332:323-327 (1988); Morrison and Oi, Adv. Immunol., 44:65-92 (1988), Verhoeyen et al., Science 239: 1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. For example, polynucleotides comprising a first Attorney Docket No.: 60846-0002W01 sequence coding for humanized immunoglobulin framework regions and a second sequence set coding for the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated in accordance with well known procedures from a variety of human cells.

[0151] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. The preferred antibodies of, and for use according to the invention include humanized and / or chimeric monoclonal antibodies.

[0152] An “antibody variant” as provided herein refers to a polypeptide capable of binding to an antigen and including one or more structural domains of an antibody or fragment thereof. Non-limiting examples of antibody variants include single-domain antibodies or nanobodies, affibodies (polypeptides smaller than monoclonal antibodies (e.g., about 6kDA) and capable of binding antigens with high affinity and imitating monoclonal antibodies, monospecific Fab?, bispecific Fab?, trispecific Fabs, monovalent IgGs, scFv, bispecific diabodies, trispecific triabodies, scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptibodies. A “nanobody” or “single domain antibody” as described herein is commonly well known in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. A “peptibody” as provided herein refers to a peptide moiety attached (through a covalent or non-covalent linker) to the Fc domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. A general description of antibodies from camelids and the variable regions thereof and methods for their production, isolation, and use may be found in references WO97 / 49805 and WO 97 / 49805 which are incorporated by reference herein in their entirety and for all purposes. Likewise, antibodies from cartilaginous fish and the variable regions thereof and methods for their production, isolation, and use may be found in W02005 / 118629, which is incorporated by reference herein in its entirety and for all purposes. Attorney Docket No.: 60846-0002W01

[0153] A “single domain antibody” as provided herein refers to an antibody fragment including a single monomeric variable antibody domain. Like a whole antibody, a single domain antibody is able to bind selectively to a specific antigen. The molecular weight of a single domain antibody is 12-15 kDa, single domain antibody. In embodiments, a single domain antibody is a variable heavy chain domain. In embodiments, a single domain antibody is a variable light chain domain. Non-limiting examples of single domain antibodies include camelid-derived VHH fragments and VNAR (variable immunoglobulin new antigen receptor) fragments. In embodiments, the single-domain antibody is a peptide domain of about 110 amino acids. In embodiments, the single-domain antibody includes a variable heavy chain domain. In embodiments, the single-domain antibody includes a variable light chain domain.

[0154] A single-chain variable fragment (scFv) is typically a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of 10 to about 25 amino acids. The linker may usually be rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can either connect the N- terminus of the VH with the C-terminus of the VL, or vice versa.

[0155] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Attorney Docket No.: 60846-0002W01

[0156] A "ligand" refers to an agent, e.g., a polypeptide or other molecule, capable of binding to a ligand binding domain (e.g., receptor or antibody, antibody variant, antibody region or fragment thereof).

[0157] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0158] The term "contacting" may include allowing two species to react, interact, or physically touch (e.g., bind), wherein the two species may be, for example, an antibody construct as described herein and a cancer protein. In embodiments, contacting includes, for example, allowing an antibody construct to bind to a cancer protein expressed on a cancer cell.

[0159] A "cell" as used herein, refers to a cell carrying out metabolic or other functions sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect e.g., spodoptera) and human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.

[0160] “B Cells” or “B lymphocytes” refer to their standard use in the art. B cells are lymphocytes, a type of white blood cell (leukocyte), that develops into a plasma cell (a “mature B cell”), which produces antibodies. An “immature B cell” is a cell that can develop into a mature B cell. Generally, pro-B cells undergo immunoglobulin heavy chain rearrangement to become pro B pre B cells, and further undergo immunoglobulin light chain rearrangement to become an immature B cells. Immature B cells include T1 and T2 B cells.

[0161] “T cells” or “T lymphocytes” as used herein are a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence Attorney Docket No.: 60846-0002W01 of a T-cell receptor on the cell surface. T cells include, for example, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTLs), regulatory T (Treg) cells, and T helper cells. Different types of T cells can be distinguished by use of T cell detection agents.

[0162] A “memory T cell” is a T cell that has previously encountered and responded to its cognate antigen during prior infection, encounter with cancer or previous vaccination. At a second encounter with its cognate antigen memory T cells can reproduce (divide) to mount a faster and stronger immune response than the first time the immune system responded to the pathogen.

[0163] A “regulatory T cell” or “suppressor T cell” is a lymphocyte which modulates the immune system, maintains tolerance to self-antigens, and prevents autoimmune disease.

[0164] The term “effector cell ligand” as provided herein refers to a cell surface molecule expressed on an effector cell of the immune system (e.g., a cytotoxic T cell, a helper T cell, a B cell, a natural killer cell). Upon binding of the first antibody region to the effector cell ligand expressed on the effector cell, the effector cell is activated and able to exert its function (e.g., selective killing or eradication of malignant, infected or otherwise unhealthy cells). In embodiments, the effector cell ligand is a CD3 protein. In embodiments, the effector cell ligand is a CD 16 protein. In embodiments, the effector cell ligand is a CD32 protein. In embodiments, the effector cell ligand is a NKp46 protein. The first antibody region as provided herein may be an antibody, an antibody variant, a fragment of an antibody or a fragment of an antibody variant.

[0165] The term "plasmid," "expression vector," or “viral vector” refers to a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, gene and regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids. Suitable viral vectors contemplated herein include, for example, lentiviral vectors and onco-retroviral vectors.

[0166] "Biological sample" or "sample" refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and Attorney Docket No.: 60846-0002W01 transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. In some embodiments, the sample is obtained from a human.

[0167] A "control" sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test condition, e.g., in the presence of a test compound, and compared to samples from known conditions, e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control). A control can also represent an average value gathered from a number of tests or results. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.

[0168] "Patient" or "subject in need thereof refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a composition or pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human.

[0169] The terms "disease" or "condition" refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein. In embodiments, the disease is cancer (e.g. lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma (Mantel cell lymphoma), head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma). Attorney Docket No.: 60846-0002W01

[0170] As used herein, the term "cancer" refers to all types of cancer, neoplasm or malignant tumors found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas. Exemplary cancers that may be treated with a compound, pharmaceutical composition, or method provided herein include lymphoma (e.g., Mantel cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zona lymphoma, Burkitt’s lymphoma), sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g. triple negative, ER positive, ER negative, chemotherapy resistant, herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma) , lung cancer (e.g. non-small cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration -resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia (e.g., lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia), acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma. Additional examples include, cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, esophagus, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget’s Disease of the Nipple, Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma, cancer of the pancreatic stellate cells, cancer of the hepatic stellate cells, or prostate cancer. Attorney Docket No.: 60846-0002W01

[0171] The term “leukemia” refers broadly to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood- leukemic or aleukemic (subleukemic). The P388 leukemia model is widely accepted as being predictive of in vivo anti-leukemic activity. It is believed that a compound that tests positive in the P388 assay will generally exhibit some level of anti-leukemic activity in vivo regardless of the type of leukemia being treated. Accordingly, the present application includes a method of treating leukemia, and, preferably, a method of treating acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.

[0172] As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and / or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from Attorney Docket No.: 60846-0002W01 cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.

[0173] The term "associated" or "associated with" in the context of a substance or substance activity or function associated with a disease (e.g., cancer) means that the disease is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function.

[0174] As used herein, "treatment" or "treating," or "palliating" or "ameliorating" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. Treatment includes preventing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition prior to the induction of the disease; suppressing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition after the inductive event but prior to the clinical appearance or reappearance of the disease; inhibiting the disease, that is, arresting the development of clinical symptoms by administration of a protective composition after their initial appearance; preventing re-occurring of the disease Attorney Docket No.: 60846-0002W01 and / or relieving the disease, that is, causing the regression of clinical symptoms by administration of a protective composition after their initial appearance. For example, certain methods herein treat cancer (e.g. lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma). For example, certain methods herein treat cancer by decreasing or reducing or preventing the occurrence, growth, metastasis, or progression of cancer; or treat cancer by decreasing a symptom of cancer. Symptoms of cancer (e.g. lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma) would be known or may be determined by a person of ordinary skill in the art.

[0175] As used herein the terms “treatment,” “treat,” or “treating” refers to a method of reducing the effects of one or more symptoms of a disease or condition characterized by expression of the protease or symptom of the disease or condition characterized by expression of the protease. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. Further, as used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination.

[0176] An "effective amount" is an amount sufficient to accomplish a stated purpose (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, reduce one or more symptoms of a disease or condition). An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or Attorney Docket No.: 60846-0002W01 symptoms of a disease, which could also be referred to as a "therapeutically effective amount." A "reduction" of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). A "prophylactically effective amount" of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. An "activity decreasing amount," as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme or protein relative to the absence of the antagonist. A "function disrupting amount," as used herein, refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist.

[0177] Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, for the given parameter, an effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0178] As used herein, the term "administering" means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other Attorney Docket No.: 60846-0002W01 modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By "co-administer" it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. The compounds of the invention can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation). The compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0179] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the antibodies provided herein suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.

[0180] Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized sepharose(TM), agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents ( / .< ., adjuvants). Attorney Docket No.: 60846-0002W01

[0181] Suitable formulations for rectal administration include, for example, suppositories, which consist of the packaged nucleic acid with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the compound of choice with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

[0182] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. Parenteral administration, oral administration, and intravenous administration are the preferred methods of administration. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[0183] Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.

[0184] The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The composition can, if desired, also contain other compatible therapeutic agents.

[0185] The combined administration contemplates co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Attorney Docket No.: 60846-0002W01

[0186] Effective doses of the compositions provided herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. However, a person of ordinary skill in the art would immediately recognize appropriate and / or equivalent doses looking at dosages of approved compositions for treating and preventing cancer for guidance.

[0187] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances, and the like, that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0188] The term "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

[0189] The term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration. Attorney Docket No.: 60846-0002W01

[0190] The pharmaceutical preparation is optionally in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The unit dosage form can be of a frozen dispersion.

[0191] The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In embodiments, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al -Muhammed, J. MicroencapsuL 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46: 1576-1587, 1989). The compositions of the present invention can also be delivered as nanoparticles.

[0192] HEAVY CHAIN SINGLE VARIABLE DOMAIN (VHH) ANTIBODIES

[0193] Monoclonal and recombinant antibodies are important tools in medicine and biotechnology. Like all mammals, camelids (e.g., alpacas and llamas) can produce Attorney Docket No.: 60846-0002W01 conventional antibodies made of two heavy chains and two light chains bound together with disulfide bonds in a Y shape (e.g., IgGl). However, they also produce a unique classes of IgG, also known as heavy chain IgG. These antibodies are made of only two heavy chains, which lack the CHI region but still bear an antigen-binding domain at their N-terminus called VHH (or nanobody). Conventional immunoglobulins require the association of variable regions from both heavy and light chains to allow a high diversity of antigenantibody interactions. Although isolated heavy and light chains still show this capacity, they exhibit very low affinity when compared to paired heavy and light chains. The unique feature of heavy chain IgG is the capacity of their monomeric antigen binding regions to bind antigens with specificity, affinity and especially diversity that are comparable to conventional antibodies without the need of pairing with another region. This feature is mainly due to a couple of major variations within the amino acid sequence of the variable region of the two heavy chains, which induce deep conformational changes when compared to conventional Ig. Major substitutions in the variable regions prevent the light chains from binding to the heavy chains, but also prevent unbound heavy chains from being recycled by the Immunoglobulin Binding Protein (IBP).

[0194] The single variable domain of these antibodies (designated VHH, sdAb, or nanobody) is the smallest antigen-binding domain generated by adaptive immune systems. The third Complementarity Determining Region (CDR3) of the variable region of these antibodies has been found to be twice as long as the conventional ones. This results in an increased interaction surface with the antigen as well as an increased diversity of antigen-antibody interactions, which compensates the absence of the light chains. With a long complementarity-determining region 3 (CDR3), VHHs can extend into crevices on proteins that are not accessible to conventional antibodies, including functionally interesting sites such as the active site of an enzyme or the receptor-binding canyon on a virus surface. Moreover, an additional cysteine residue allow the structure to be more stable, thus increasing the strength of the interaction.

[0195] VHHs offer numerous other advantages compared to conventional antibodies carrying variable domains (VH and VL) of conventional antibodies, including higher stability, solubility, expression yields, and refolding capacity, as well as better in vivo tissue penetration. Moreover, in contrast to the VH domains of conventional antibodies, VHHs do not display an intrinsic tendency to bind to light chains. This facilitates the induction of heavy Attorney Docket No.: 60846-0002W01 chain antibodies in the presence of a functional light chain loci. Further, since VHHs do not bind to VL domains, it is much easier to reformat VHHs into bispecific antibody constructs than constructs containing conventional VH-VL pairs or single domains based on VH domains.

[0196] The disclosure provides e.g., anti-DLL3 antibodies, the modified antibodies thereof, the camelid antibodies thereof, the chimeric antibodies thereof, and the humanized antibodies thereof.

[0197] The CDR sequences for VHH1, and VHH1 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 13, 14, and 15, respectively, as defined by Kabat definition. The CDRs can also be defined by Chothia system. Under the Chothia definition, the CDR sequences for VHH1, and VHH1 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 103, 104, and 105, respectively.

[0198] The CDR sequences for VHH2, and VHH2 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 16, 17, and 18, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH2, and VHH2 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 106, 107, and 108, respectively.

[0199] The CDR sequences for VHH3, and VHH3 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 19, 20, and 21, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH3, and VHH3 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 109, 110, and 111, respectively..

[0200] The CDR sequences for VHH4, and VHH4 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 22, 23, and 24, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH4, and VHH4 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 112, 113, and 114, respectively..

[0201] The CDR sequences for VHH5, and VHH5 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 25, 26, and 27, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for Attorney Docket No.: 60846-0002W01

[0202] VHH5, and VHH5 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 115, 116, and 117, respectively..

[0203] The CDR sequences for VHH6, and VHH6 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 28, 29, and 30, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH6, and VHH6 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 118, 119, and 120, respectively..

[0204] The CDR sequences for VHH7, and VHH7 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 31, 32, and 33, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH7, and VHH7 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 121, 122, and 123, respectively..

[0205] The CDR sequences for VHH8, and VHH8 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 34, 35, and 36, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH8, and VHH8 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 124, 125, and 126, respectively..

[0206] The CDR sequences for VHH9, and VHH9 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 37, 38, and 39, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH9, and VHH9 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 127, 128, and 129, respectively..

[0207] The CDR sequences for VHHIO, and VHH10 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 40, 41, and 42, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHHIO, and VHH10 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 130, 131, and 132, respectively.

[0208] The CDR sequences for VHH11, and VHH11 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 43, 44, and 45, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH11, and VHH11 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 133, 134, and 135, respectively.. Attorney Docket No.: 60846-0002W01

[0209] The CDR sequences for VHH12, and VHH12 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 46, 47, and 48, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH12, and VHH12 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 136, 137, and 138, respectively.

[0210] The CDR sequences for VHH13, and VHH13 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 49, 50, and 51, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH13, and VHH13 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 139, 140, and 141, respectively..

[0211] The CDR sequences for VHH14, and VHH14 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 52, 53, and 54, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH14, and VHH14 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 142, 143, and 144, respectively..

[0212] The CDR sequences for VHH15, and VHH15 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 55, 56, and 57, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH15, and VHH15 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 145, 146, and 147, respectively.

[0213] The CDR sequences for VHH16, and VHH16 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 58, 59, and 60, respectively as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH16, and VHH16 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 148, 149, and 150, respectively.

[0214] The amino acid sequence for the VHH domain of VHH1 antibody is set forth in SEQ ID NO: 193. The amino acid sequence for the VHH domain of VHH2 antibody is set forth in SEQ ID NO: 194. The amino acid sequence for the VHH domain of VHH3 antibody is set forth in SEQ ID NO: 195. The amino acid sequence for the VHH domain of VHH4 antibody is set forth in SEQ ID NO: 196. The amino acid sequence for the VHH domain of VHH5 antibody is set forth in SEQ ID NO: 197. The amino acid sequence for the VHH domain of VHH6 antibody is set forth in SEQ ID NO: 198. The amino acid sequence for the VHH Attorney Docket No.: 60846-0002W01 domain of VHH7 antibody is set forth in SEQ ID NO: 199. The amino acid sequence for the VHH domain of VHH8 antibody is set forth in SEQ ID NO: 200. The amino acid sequence for the VHH domain of VHH9 antibody is set forth in SEQ ID NO: 201. The amino acid sequence for the VHH domain of VHH10 antibody is set forth in SEQ ID NO: 202. The amino acid sequence for the VHH domain of VHH11 antibody is set forth in SEQ ID NO: 203. The amino acid sequence for the VHH domain of VHH12 antibody is set forth in SEQ ID NO: 204. The amino acid sequence for the VHH domain of VHH13 antibody is set forth in SEQ ID NO: 205. The amino acid sequence for the VHH domain of VHH14 antibody is set forth in SEQ ID NO: 206. The amino acid sequence for the VHH domain of VHH15 antibody is set forth in SEQ ID NO: 207. The amino acid sequence for the VHH domain of VHH16 antibody is set forth in SEQ ID NO: 208. The amino acid sequence for the VHH domain of VHH15 antibody is set forth in SEQ ID NO: 571. The amino acid sequence for the VHH domain of VHH15 antibody is set forth in SEQ ID NO: 572.

[0215] The amino acid sequences for various modified or humanized VHH are also provided. As there are different ways to modify or humanize a camelid antibody (e.g., a sequence can be modified with different amino acid substitutions), the VHH domain of an antibody can have more than one version of humanized sequences. In some embodiments, the humanized VHH domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any sequence of SEQ ID NOs: 193-208, 571, and 572.

[0216] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three VHH domain CDRs selected from the group of SEQ ID NOs: 13-15, SEQ ID NOs: 16-18, SEQ ID NOs: 19-21, SEQ ID NOs: 22-24, SEQ ID NOs: 25-27, SEQ ID NOs: 28-30, SEQ ID NOs: 31-33, SEQ ID NOs: 34-36, SEQ ID NOs: 37-39, SEQ ID NOs: 40-42, SEQ ID NOs: 43-45, SEQ ID NOs: 46-48, SEQ ID NOs: 49-51, SEQ ID NOs: 52-54, SEQ ID NOs: 55-57, SEQ ID NOs: 58-60, SEQ ID NOs: 103-105, SEQ ID NOs: 106-108, SEQ ID NOs: 109-111, SEQ ID NOs: 112-114, SEQ ID NOs: 115-117, SEQ ID NOs: 118-120, SEQ ID NOs: 121-123, SEQ ID NOs: 124- 126, SEQ ID NOs: 127-129, SEQ ID NOs: 130-132, SEQ ID NOs: 133-135, SEQ ID NOs: 136-138, SEQ ID NOs: 139-141, SEQ ID NOs: 142-144, SEQ ID NOs: 145-147, and SEQ ID NOs: 148-150.

[0217] The disclosure also provides antibodies or antigen-binding fragments thereof that bind to DLL3 (e.g., human DLL3). The antibodies or antigen-binding fragments thereof contain a Attorney Docket No.: 60846-0002W01 heavy chain single variable region (VHH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH sequence. In some embodiments, the selected VHH sequence is SEQ ID NO: 193. In some embodiments, the selected VHH sequence is SEQ ID NO: 194. In some embodiments, the selected VHH sequence is SEQ ID NO: 195. In some embodiments, the selected VHH sequence is SEQ ID NO: 196. In some embodiments, the selected VHH sequence is SEQ ID NO: 197. In some embodiments, the selected VHH sequence is SEQ ID NO: 198. In some embodiments, the selected VHH sequence is SEQ ID NO: 199. In some embodiments, the selected VHH sequence is SEQ ID NO: 200. In some embodiments, the selected VHH sequence is SEQ ID NO: 201. In some embodiments, the selected VHH sequence is SEQ ID NO: 202. In some embodiments, the selected VHH sequence is SEQ ID NO: 203. In some embodiments, the selected VHH sequence is SEQ ID NO: 204. In some embodiments, the selected VHH sequence is SEQ ID NO: 205. In some embodiments, the selected VHH sequence is SEQ ID NO: 206. In some embodiments, the selected VHH sequence is SEQ ID NO: 207. In some embodiments, the selected VHH sequence is SEQ ID NO: 208. In some embodiments, the selected VHH sequence is SEQ ID NO: 571. In some embodiments, the selected VHH sequence is SEQ ID NO: 572.

[0218] In some embodiments, the antibody or antigen-binding fragment thereof described herein comprises a heavy chain single variable domain (VHH) CDR1 selected from SEQ ID NOs: 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, and 148. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR2 selected from SEQ ID NOs: 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, and 149. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR3 selected from SEQ ID NOs: 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, and 150.

[0219] In some embodiments, the VHH CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, and 148. In some embodiments, the VHH CDR2 region Attorney Docket No.: 60846-0002W01 comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, and 149. In some embodiments, the VHH CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, and 150. In some embodiments, the VHH described herein comprises any one of the VHH CDR1 regions described herein, any one of the VHH CDR2 regions described herein, and any one of the VHH CDR3 regions described herein.

[0220] The disclosure also provides e.g., anti-CD3 antibodies, the modified antibodies thereof, the camelid antibodies thereof, the chimeric antibodies thereof, and the humanized antibodies thereof.

[0221] The CDR sequences for VHH17, and VHH17 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 61, 62, and 63, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH17, and VHH17 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 151, 152, and 153, respectively.

[0222] The CDR sequences for VHH18, and VHH18 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 64, 65, and 66, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH18, and VHH18 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 154, 155, and 156, respectively.

[0223] The CDR sequences for VHH19, and VHH19 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 67, 68, and 69, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH19, and VHH19 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 157, 158, and 159, respectively.

[0224] The CDR sequences for VHH20, and VHH20 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 70, 71, and 72, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH20, and VHH20 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 160, 161, and 162, respectively. Attorney Docket No.: 60846-0002W01

[0225] The CDR sequences for VHH21, and VHH21 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 73, 74, and 75, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH21, and VHH21 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 163, 164, and 165, respectively.

[0226] The CDR sequences for VHH22, and VHH22 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 76, 77, and 78, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH22, and VHH22 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 166, 167, and 168, respectively.

[0227] The CDR sequences for VHH23, and VHH23 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 79, 80, and 81, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH23, and VHH23 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 169, 170, and 171, respectively.

[0228] The CDR sequences for VHH24, and VHH24 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 82, 83, and 84, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH24, and VHH24 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 172, 173, and 174, respectively.

[0229] The CDR sequences for VHH25, and VHH25 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 85, 86, and 87, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH25, and VHH25 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 175, 176, and 177, respectively.

[0230] The CDR sequences for VHH26, and VHH26 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 88, 89, and 90, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH26, and VHH26 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 178, 179, and 180, respectively.

[0231] The CDR sequences for VHH27, and VHH27 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 91, 92, and 93, Attorney Docket No.: 60846-0002W01 respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH27, and VHH27 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 181, 182, and 183, respectively.

[0232] The CDR sequences for VHH28, and VHH28 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 94, 95, and 96, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH28, and VHH28 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 184, 185, and 186, respectively.

[0233] The CDR sequences for VHH29, and VHH29 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 97, 98, and 99, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH29, and VHH29 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 187, 188, and 189, respectively.

[0234] The CDR sequences for VHH30, and VHH30 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 100, 101, and 102, respectively, as defined by Kabat definition. Under Chothia definition, the CDR sequences for VHH30, and VHH30 derived antibodies (e.g., humanized antibodies) include CDRs of the VHH domain as set forth in SEQ ID NOs: 190, 191, and 192, respectively.

[0235] The amino acid sequence for the VHH domain of VHH17 antibody is set forth in SEQ ID NO: 209. The amino acid sequence for the VHH domain of VHH18 antibody is set forth in SEQ ID NO: 210. The amino acid sequence for the VHH domain of VHH19 antibody is set forth in SEQ ID NO: 211. The amino acid sequence for the VHH domain of VHH20 antibody is set forth in SEQ ID NO: 212. The amino acid sequence for the VHH domain of VHH21 antibody is set forth in SEQ ID NO: 213. The amino acid sequence for the VHH domain of VHH22 antibody is set forth in SEQ ID NO: 214. The amino acid sequence for the VHH domain of VHH23 antibody is set forth in SEQ ID NO: 215. The amino acid sequence for the VHH domain of VHH24 antibody is set forth in SEQ ID NO: 216. The amino acid sequence for the VHH domain of VHH25 antibody is set forth in SEQ ID NO: 217. The amino acid sequence for the VHH domain of VHH26 antibody is set forth in SEQ ID NO: 218. The amino acid sequence for the VHH domain of VHH27 antibody is set forth in SEQ ID NO: 219. The amino acid sequence for the VHH domain of VHH28 antibody is set forth in SEQ ID NO: 220. The amino acid sequence for the VHH domain of VHH29 antibody is set forth Attorney Docket No.: 60846-0002W01 in SEQ ID NO: 221. The amino acid sequence for the VHH domain of VHH30 antibody is set forth in SEQ ID NO: 222.

[0236] The amino acid sequences for various modified or humanized VHH are also provided. As there are different ways to modify or humanize a camelid antibody (e.g., a sequence can be modified with different amino acid substitutions), the VHH domain of an antibody can have more than one version of humanized sequences. In some embodiments, the humanized VHH domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any sequence of SEQ ID NOs: 209-222.

[0237] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three VHH domain CDRs selected from the group of SEQ ID NOs: 61-63, SEQ ID NOs: 64-66, SEQ ID NOs: 67-69, SEQ ID NOs: 70-72, SEQ ID NOs: 73-75, SEQ ID NOs: 76-78, SEQ ID NOs: 79-81, SEQ ID NOs: 82-84, SEQ ID NOs: 85-87, SEQ ID NOs: 88-90, SEQ ID NOs: 91-93, SEQ ID NOs: 94-96, SEQ ID NOs: 97-99, SEQ ID NOs: 100-102, SEQ ID NOs: 151-153, SEQ ID NOs: 154-156, SEQ ID NOs: 157-159, SEQ ID NOs: 160-162, SEQ ID NOs: 163-165, SEQ ID NOs: 166- 168, SEQ ID NOs: 169-171, SEQ ID NOs: 172-174, SEQ ID NOs: 175-177, SEQ ID NOs: 178-180, SEQ ID NOs: 181-183, SEQ ID NOs: 184-186, SEQ ID NOs: 187-189, and SEQ ID NOs: 190-192.

[0238] The disclosure also provides antibodies or antigen-binding fragments thereof that bind to CD3 (e.g., human CD3). The antibodies or antigen-binding fragments thereof contain a heavy chain single variable region (VHH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH sequence. In some embodiments, the selected VHH sequence is SEQ ID NO: 209. In some embodiments, the selected VHH sequence is SEQ ID NO: 210. In some embodiments, the selected VHH sequence is SEQ ID NO: 211. In some embodiments, the selected VHH sequence is SEQ ID NO: 212. In some embodiments, the selected VHH sequence is SEQ ID NO: 213. In some embodiments, the selected VHH sequence is SEQ ID NO: 214. In some embodiments, the selected VHH sequence is SEQ ID NO: 215. In some embodiments, the selected VHH sequence is SEQ ID NO: 216. In some embodiments, the selected VHH sequence is SEQ ID NO: 217. In some embodiments, the selected VHH sequence is SEQ ID NO: 218. In some embodiments, the selected VHH sequence is SEQ ID NO: 219. In some embodiments, the selected VHH sequence is SEQ ID NO: 220. In some embodiments, the selected VHH Attorney Docket No.: 60846-0002W01 sequence is SEQ ID NO: 221. In some embodiments, the selected VHH sequence is SEQ ID NO: 222.

[0239] In some embodiments, the antibody or antigen-binding fragment thereof described herein comprises a heavy chain single variable domain (VHH) CDR1 selected from SEQ ID NOs: 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, and 190. In some embodiments, the antibody or antigenbinding fragment thereof comprises a heavy chain single variable domain (VHH) CDR2 selected from SEQ ID NOs: 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, and 191. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR3 selected from SEQ ID NOs: 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, and 192.

[0240] In some embodiments, the VHH CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, and 190. In some embodiments, the VHH CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, and 191. In some embodiments, the VHH CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, and 192. In some embodiments, the VHH described herein comprises any one of the VHH CDR1 regions described herein, any one of the VHH CDR2 regions described herein, and any one of the VHH CDR3 regions described herein.

[0241] In some embodiments, the antibodies can have a heavy chain single variable domain (VHH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH Attorney Docket No.: 60846-0002W01

[0242] CDR3 amino acid sequence. The selected VHH CDRs 1, 2, 3 amino acid sequences is shown in FIG. 16 and FIG. 17

[0243] In some embodiments, provided herein are antibodies having a heavy chain single variable domain (VHH) comprising VHH CDRs 1, 2, 3, wherein the VHH CDRs 1, 2, 3 are identical to the CDRs 1, 2, 3 of the VHHs shown in FIG. 18.

[0244] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain single variable domain (VHH) containing one, two, or three of VHH CDR1 with zero, one or two amino acid insertions, deletions, or substitutions; VHH CDR2 with zero, one or two amino acid insertions, deletions, or substitutions; VHH CDR3 with zero, one or two amino acid insertions, deletions, or substitutions, wherein VHH CDR1, VHH CDR2, and VHH CDR3 are selected from the CDRs in FIG. 16 and FIG. 17. The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence. In some embodiments, the CDR is determined based on Chothia numbering scheme. In some embodiments, the CDR is determined based on Kabat numbering scheme. In some embodiments, the CDR is determined based on IMGT numbering scheme. In some embodiments, the CDR is determined based on contact definition. In some embodiments, the CDR is determined based on a combination of numbering schemes. Details of different numbering schemes can be found, e.g., in Dondelinger, M., et al. "Understanding the significance and implications of antibody numbering and antigen-binding surface / residue definition." Frontiers in Immunology 9 (2018): 2278, which is incorporated herein by reference in its entirety.

[0245] The disclosure also provides nucleic acids comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain single variable domain (e.g., any of the VHHs described herein). In some embodiments, the VHH comprises CDRs as shown in FIG. 16 and FIG. 17, or has sequences as shown in FIG. 18.

[0246] ANTIBODIES AND ANTIGEN-BINDING FRAGMENTS

[0247] The disclosure provides, e.g., anti-CD3 antibodies, the modified antibodies thereof, including, e.g., chimeric antibodies, humanized antibodies, and human antibodies. The disclosure also provides antigen-binding fragments (e.g., scFvs) of the anti-CD3 antibodies.

[0248] The CDR sequences for ScFvl, and ScFvl derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 223, 224, 225, Attorney Docket No.: 60846-0002W01 and CDRs of the light chain variable domain, SEQ ID NOs: 226, 227, 228, as defined by Kabat definition. The CDRs can also be defined by Chothia system. Under the Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 355, 356, 357, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 358, 359, 360.

[0249] The CDR sequences for ScFv2, and ScFv2 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 229, 230, 231, and CDRs of the light chain variable domain, SEQ ID NOs: 232, 233, 234, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 361, 362, 363, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 364, 365, 366.

[0250] The CDR sequences for ScFv3, and ScFv3 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 235, 236, 237, and CDRs of the light chain variable domain, SEQ ID NOs: 238, 239, 240, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 367, 368, 369, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 370, 371, 372.

[0251] The CDR sequences for ScFv4, and ScFv4 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 241, 242, 243, and CDRs of the light chain variable domain, SEQ ID NOs: 244, 245, 246, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 373, 374, 375, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 376, 377, 378.

[0252] The CDR sequences for ScFv5, and ScFv5 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 247, 248, 249, and CDRs of the light chain variable domain, SEQ ID NOs: 250, 251, 252, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 379, 380, 381, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 382, 383, 384.

[0253] The CDR sequences for ScFv6, and ScFv6 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 253, 254, 255, and CDRs of the light chain variable domain, SEQ ID NOs: 256, 257, 258, as defined by Kabat definition. Under Chothia Attorney Docket No.: 60846-0002W01 definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 385, 386, 387, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 388, 389, 390.

[0254] The CDR sequences for ScFv7, and ScFv7 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 259, 260, 261, and CDRs of the light chain variable domain, SEQ ID NOs: 262, 263, 264, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 391, 392, 393, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 394, 395, 396.

[0255] The CDR sequences for ScFv8, and ScFv8 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 265, 266, 267, and CDRs of the light chain variable domain, SEQ ID NOs: 268, 269, 270, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 397, 398, 399, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 400, 401, 402.

[0256] The CDR sequences for ScFv9, and ScFv9 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 271, 272, 273, and CDRs of the light chain variable domain, SEQ ID NOs: 274, 275, 276, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 403, 404, 405, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 406, 407, 408.

[0257] The CDR sequences for ScFvlO, and ScFvlO derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 277, 278, 279, and CDRs of the light chain variable domain, SEQ ID NOs: 280, 281, 282, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 409, 410, 411, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 412, 413, 414.

[0258] The CDR sequences for ScFvll, and ScFvll derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 283, 284, 285, and CDRs of the light chain variable domain, SEQ ID NOs: 286, 287, 288, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID Attorney Docket No.: 60846-0002W01

[0259] NOs: 415, 416, 417, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 418, 419, 420.

[0260] The CDR sequences for ScFvl2, and ScFvl2 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 289, 290, 291, and CDRs of the light chain variable domain, SEQ ID NOs: 292, 293, 294, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 421, 422, 423, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 424, 425, 426.

[0261] The CDR sequences for ScFvl3, and ScFvl3 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 295, 296, 297, and CDRs of the light chain variable domain, SEQ ID NOs: 298, 299, 300, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 427, 428, 429, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 430, 431, 432.

[0262] The CDR sequences for ScFvl4, and ScFvl4 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 301, 302, 303, and CDRs of the light chain variable domain, SEQ ID NOs: 304, 305, 306, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 433, 434, 435, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 436, 437, 438.

[0263] The CDR sequences for ScFvl5, and ScFvl5 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 307, 308, 309, and CDRs of the light chain variable domain, SEQ ID NOs: 310, 311, 312, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 439, 440, 441, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 442, 443, 444.

[0264] The CDR sequences for ScFvl6, and ScFvl6 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 313, 314, 315, and CDRs of the light chain variable domain, SEQ ID NOs: 316, 317, 318, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 445, 446, 447, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 448, 449, 450. Attorney Docket No.: 60846-0002W01

[0265] The amino acid sequence for the heavy chain variable region of ScFvl, and ScFvl derived antibodies is set forth in SEQ ID NO: 489. The amino acid sequence for the light chain variable region of ScFvl, and ScFvl derived antibodies is set forth in SEQ ID NO: 488.

[0266] The amino acid sequence for the heavy chain variable region of ScFv2, and ScFv2 derived antibodies is set forth in SEQ ID NO: 492. The amino acid sequence for the light chain variable region of ScFv2, and ScFv2 derived antibodies is set forth in SEQ ID NO: 491.

[0267] The amino acid sequence for the heavy chain variable region of ScFv3, and ScFv3 derived antibodies is set forth in SEQ ID NO: 495. The amino acid sequence for the light chain variable region of ScFv3, and ScFv3 derived antibodies is set forth in SEQ ID NO: 494.

[0268] The amino acid sequence for the heavy chain variable region of ScFv4, and ScFv4 derived antibodies is set forth in SEQ ID NO: 498. The amino acid sequence for the light chain variable region of ScFv4, and ScFv4 derived antibodies is set forth in SEQ ID NO: 497.

[0269] The amino acid sequence for the heavy chain variable region of ScFv5, and ScFv5 derived antibodies is set forth in SEQ ID NO: 501. The amino acid sequence for the light chain variable region of ScFv5, and ScFv5 derived antibodies is set forth in SEQ ID NO: 500.

[0270] The amino acid sequence for the heavy chain variable region of ScFv6, and ScFv6 derived antibodies is set forth in SEQ ID NO: 504. The amino acid sequence for the light chain variable region of ScFv6, and ScFv6 derived antibodies is set forth in SEQ ID NO: 503.

[0271] The amino acid sequence for the heavy chain variable region of ScFv7, and ScFv7 derived antibodies is set forth in SEQ ID NO: 507. The amino acid sequence for the light chain variable region of ScFv7, and ScFv7 derived antibodies is set forth in SEQ ID NO: 506.

[0272] The amino acid sequence for the heavy chain variable region of ScFv8, and ScFv8 derived antibodies is set forth in SEQ ID NO: 510. The amino acid sequence for the light chain variable region of ScFv8, and ScFv8 derived antibodies is set forth in SEQ ID NO: 509.

[0273] The amino acid sequence for the heavy chain variable region of ScFv9, and ScFv9 derived antibodies is set forth in SEQ ID NO: 513. The amino acid sequence for the light chain variable region of ScFv9, and ScFv9 derived antibodies is set forth in SEQ ID NO: 512.

[0274] The amino acid sequence for the heavy chain variable region of ScFvlO, and ScFvlO derived antibodies is set forth in SEQ ID NO: 516. The amino acid sequence for the light chain variable region of ScFvlO, and ScFvlO derived antibodies is set forth in SEQ ID NO: 515. Attorney Docket No.: 60846-0002W01

[0275] The amino acid sequence for the heavy chain variable region of ScFvll, and ScFvll derived antibodies is set forth in SEQ ID NO: 519. The amino acid sequence for the light chain variable region of ScFvll, and ScFvll derived antibodies is set forth in SEQ ID NO: 518.

[0276] The amino acid sequence for the heavy chain variable region of ScFvl2, and ScFvl2 derived antibodies is set forth in SEQ ID NO: 522. The amino acid sequence for the light chain variable region of ScFvl2, and ScFvl2 derived antibodies is set forth in SEQ ID NO: 521.

[0277] The amino acid sequence for the heavy chain variable region of ScFvl3, and ScFvl3 derived antibodies is set forth in SEQ ID NO: 525. The amino acid sequence for the light chain variable region of ScFvl3, and ScFvl3 derived antibodies is set forth in SEQ ID NO: 524.

[0278] The amino acid sequence for the heavy chain variable region of ScFvl4, and ScFvl4 derived antibodies is set forth in SEQ ID NO: 528. The amino acid sequence for the light chain variable region of ScFvl4, and ScFvl4 derived antibodies is set forth in SEQ ID NO: 527.

[0279] The amino acid sequence for the heavy chain variable region of ScFvl5, and ScFvl5 derived antibodies is set forth in SEQ ID NO: 531. The amino acid sequence for the light chain variable region of ScFvl5, and ScFvl5 derived antibodies is set forth in SEQ ID NO: 530.

[0280] The amino acid sequence for the heavy chain variable region of ScFvl6, and ScFvl6 derived antibodies is set forth in SEQ ID NO: 534. The amino acid sequence for the light chain variable region of ScFvl6, and ScFvl6 derived antibodies is set forth in SEQ ID NO: 533.

[0281] The amino acid sequence for the heavy chain variable region of ScFv23, and ScFv23 derived antibodies is set forth in SEQ ID NO: 575. The amino acid sequence for the light chain variable region of ScFv23, and ScFv23 derived antibodies is set forth in SEQ ID NO: 574.

[0282] The amino acid sequence for the heavy chain variable region of ScFv24, and ScFv24 derived antibodies is set forth in SEQ ID NO: 578. The amino acid sequence for the light chain variable region of ScFv24, and ScFv24 derived antibodies is set forth in SEQ ID NO: 577. Attorney Docket No.: 60846-0002W01

[0283] The amino acid sequences for heavy chain variable regions and light variable regions of the modified antibodies are also provided. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, 528, 531, 534, 575, or 578. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, 527, 530, 533, 574, or 577. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, and together they bind to CD3.

[0284] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 223-225, SEQ ID NOs: 229-231, SEQ ID NOs: 235-237, SEQ ID NOs: 241-243, SEQ ID NOs: 247-249, SEQ ID NOs: 253-255, SEQ ID NOs: 259-261, SEQ ID NOs: 265-267, SEQ ID NOs: 271-273, SEQ ID NOs: 277-279, SEQ ID NOs: 283-285, SEQ ID NOs: 289-291, SEQ ID NOs: 295-297, SEQ ID NOs: 301- 303, SEQ ID NOs: 307-309, SEQ ID NOs: 313-315, SEQ ID NOs: 355-357, SEQ ID NOs: 361-363, SEQ ID NOs: 367-369, SEQ ID NOs: 373-375, SEQ ID NOs: 379-381, SEQ ID NOs: 385-387, SEQ ID NOs: 391-393, SEQ ID NOs: 397-399, SEQ ID NOs: 403-405, SEQ ID NOs: 409-411, SEQ ID NOs: 415-417, SEQ ID NOs: 421-423, SEQ ID NOs: 427-429, SEQ ID NOs: 433-435, SEQ ID NOs: 439-441, and SEQ ID NOs: 445-447; and / or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 226-228, SEQ ID NOs: 232-234, SEQ ID NOs: 238-240, SEQ ID NOs: 244-246, SEQ ID NOs: 250- 252, SEQ ID NOs: 256-258, SEQ ID NOs: 262-264, SEQ ID NOs: 268-270, SEQ ID NOs: 274-276, SEQ ID NOs: 280-282, SEQ ID NOs: 286-288, SEQ ID NOs: 292-294, SEQ ID NOs: 298-300, SEQ ID NOs: 304-306, SEQ ID NOs: 310-312, SEQ ID NOs: 316-318, SEQ ID NOs: 358-360, SEQ ID NOs: 364-366, SEQ ID NOs: 370-372, SEQ ID NOs: 376-378, SEQ ID NOs: 382-384, SEQ ID NOs: 388-390, SEQ ID NOs: 394-396, SEQ ID NOs: 400- 402, SEQ ID NOs: 406-408, SEQ ID NOs: 412-414, SEQ ID NOs: 418-420, SEQ ID NOs: 424-426, SEQ ID NOs: 430-432, SEQ ID NOs: 436-438, SEQ ID NOs: 442-444, and SEQ ID NOs: 448-450.

[0285] The disclosure also provides antibodies or antigen-binding fragments thereof that bind to CD3. The antibodies or antigen-binding fragments thereof contain a heavy chain variable Attorney Docket No.: 60846-0002W01 region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 489, and the selected VL sequence is SEQ ID NO: 488. In some embodiments, the selected VH sequence is SEQ ID NO: 492, and the selected VL sequence is SEQ ID NO: 491. In some embodiments, the selected VH sequence is SEQ ID NO: 495, and the selected VL sequence is SEQ ID NO: 494. In some embodiments, the selected VH sequence is SEQ ID NO: 498, and the selected VL sequence is SEQ ID NO: 497. In some embodiments, the selected VH sequence is SEQ ID NO: 501, and the selected VL sequence is SEQ ID NO: 500. In some embodiments, the selected VH sequence is SEQ ID NO: 504, and the selected VL sequence is SEQ ID NO: 503. In some embodiments, the selected VH sequence is SEQ ID NO: 507, and the selected VL sequence is SEQ ID NO: 506. In some embodiments, the selected VH sequence is SEQ ID NO: 510, and the selected VL sequence is SEQ ID NO: 509. In some embodiments, the selected VH sequence is SEQ ID NO: 513, and the selected VL sequence is SEQ ID NO: 512. In some embodiments, the selected VH sequence is SEQ ID NO: 516, and the selected VL sequence is SEQ ID NO: 515. In some embodiments, the selected VH sequence is SEQ ID NO: 519, and the selected VL sequence is SEQ ID NO: 518. In some embodiments, the selected VH sequence is SEQ ID NO: 522, and the selected VL sequence is SEQ ID NO: 521. In some embodiments, the selected VH sequence is SEQ ID NO: 525, and the selected VL sequence is SEQ ID NO: 524. In some embodiments, the selected VH sequence is SEQ ID NO: 528, and the selected VL sequence is SEQ ID NO: 527. In some embodiments, the selected VH sequence is SEQ ID NO: 531, and the selected VL sequence is SEQ ID NO: 530. In some embodiments, the selected VH sequence is SEQ ID NO: 534, and the selected VL sequence is SEQ ID NO: 533. In some embodiments, the selected VH sequence is SEQ ID NO: 575, and the selected VL sequence is SEQ ID NO: 574. In some embodiments, the selected VH sequence is SEQ ID NO: 578, and the selected VL sequence is SEQ ID NO: 577.

[0286] The disclosure also provides scFvs that specifically binds to CD3. In some embodiments, the scFv is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526, 529, 532, 573, or 576. Attorney Docket No.: 60846-0002W01

[0287] The disclosure also provides, e.g., anti-DLL3 antibodies, the modified antibodies thereof, including, e.g., chimeric antibodies, humanized antibodies, and human antibodies. The disclosure also provides antigen-binding fragments (e.g., scFvs) of the anti-DLL3 antibodies.

[0288] The CDR sequences for ScFvl7, and ScFvl7 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 319, 320, 321, and CDRs of the light chain variable domain, SEQ ID NOs: 322, 323, 324, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 451, 452, 453, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 454, 455, 456.

[0289] The CDR sequences for ScFvl8, and ScFvl8 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 325, 326, 327, and CDRs of the light chain variable domain, SEQ ID NOs: 328, 329, 330, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 457, 458, 459, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 460, 461, 462.

[0290] The CDR sequences for ScFvl9, and ScFvl9 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 331, 332, 333, and CDRs of the light chain variable domain, SEQ ID NOs: 334, 335, 336, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 463, 464, 465, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 466, 467, 468.

[0291] The CDR sequences for ScFv20, and ScFv20 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 337, 338, 339, and CDRs of the light chain variable domain, SEQ ID NOs: 340, 341, 342, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 469, 470, 471, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 472, 473, 474.

[0292] The CDR sequences for ScFv21, and ScFv21 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 343, 344, 345, and CDRs of the light chain variable domain, SEQ ID NOs: 346, 347, 348, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID Attorney Docket No.: 60846-0002W01

[0293] NOs: 475, 476, 477, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 478, 479, 480.

[0294] The CDR sequences for ScFv22, and ScFv22 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 349, 350, 351, and CDRs of the light chain variable domain, SEQ ID NOs: 352, 353, 354, as defined by Kabat definition. Under Chothia definition, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 481, 482, 483, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 484, 485, 486.

[0295] The amino acid sequence for the heavy chain variable region of ScFvl7, and ScFvl7 derived antibodies is set forth in SEQ ID NO: 537. The amino acid sequence for the light chain variable region of ScFvl7, and ScFvl7 derived antibodies is set forth in SEQ ID NO: 536.

[0296] The amino acid sequence for the heavy chain variable region of ScFvl8, and ScFvl8 derived antibodies is set forth in SEQ ID NO: 540. The amino acid sequence for the light chain variable region of ScFvl8, and ScFvl8 derived antibodies is set forth in SEQ ID NO: 539.

[0297] The amino acid sequence for the heavy chain variable region of ScFvl9, and ScFvl9 derived antibodies is set forth in SEQ ID NO: 543. The amino acid sequence for the light chain variable region of ScFvl9, and ScFvl9 derived antibodies is set forth in SEQ ID NO: 542.

[0298] The amino acid sequence for the heavy chain variable region of ScFv20, and ScFv20 derived antibodies is set forth in SEQ ID NO: 546. The amino acid sequence for the light chain variable region of ScFv20, and ScFv20 derived antibodies is set forth in SEQ ID NO: 545.

[0299] The amino acid sequence for the heavy chain variable region of ScFv21, and ScFv21 derived antibodies is set forth in SEQ ID NO: 549. The amino acid sequence for the light chain variable region of ScFv21, and ScFv21 derived antibodies is set forth in SEQ ID NO: 548.

[0300] The amino acid sequence for the heavy chain variable region of ScFv22, and ScFv22 derived antibodies is set forth in SEQ ID NO: 552. The amino acid sequence for the light chain variable region of ScFv22, and ScFv22 derived antibodies is set forth in SEQ ID NO: 551. Attorney Docket No.: 60846-0002W01

[0301] The amino acid sequences for heavy chain variable regions and light variable regions of the modified antibodies are also provided. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 537, 540, 543, 546, 549, or 552. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 536, 539, 542, 545, 548, or 551. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, and together they bind to DLL3.

[0302] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 319-321, SEQ ID NOs: 325-327, SEQ ID NOs: 331-333, SEQ ID NOs: 337-339, SEQ ID NOs: 343-345, SEQ ID NOs: 349-351, SEQ ID NOs: 451-453, SEQ ID NOs: 457-459, SEQ ID NOs: 463-465, SEQ ID NOs: 469-471, SEQ ID NOs: 475-477, and SEQ ID NOs: 481-483; and / or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 322-324, SEQ ID NOs: 328- 330, SEQ ID NOs: 334-336, SEQ ID NOs: 340-342, SEQ ID NOs: 346-348, SEQ ID NOs: 352-354, SEQ ID NOs: 454-456, SEQ ID NOs: 460-462, SEQ ID NOs: 466-468, SEQ ID NOs: 472-474, SEQ ID NOs: 478-480, and SEQ ID NOs: 484-486.

[0303] The disclosure also provides antibodies or antigen-binding fragments thereof that bind to DLL3. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 537, and the selected VL sequence is SEQ ID NO: 536. In some embodiments, the selected VH sequence is SEQ ID NO: 540, and the selected VL sequence is SEQ ID NO: 539. In some embodiments, the selected VH sequence is SEQ ID NO: 543, and the selected VL sequence is SEQ ID NO: 542. In some embodiments, the selected VH sequence is SEQ ID NO: 546, and the selected VL sequence is SEQ ID NO: 545. In some embodiments, the selected VH sequence is SEQ ID NO: 549, and the selected VL sequence is SEQ ID NO: 548. In some embodiments, the selected VH sequence is SEQ ID NO: 552, and the selected VL sequence is SEQ ID NO: 551. Attorney Docket No.: 60846-0002W01

[0304] The disclosure also provides scFvs that specifically binds to DLL3. In some embodiments, the scFv is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 535, 538, 541, 544, 547, or 550.

[0305] In some embodiments, the antibodies can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence. In some embodiments, the antibodies can have a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. The selected VH CDRs 1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 19 (CDRs under Kabat definition), and FIG. 20 (CDRs under Chothia definition).

[0306] In some embodiments, provided herein are antibodies having a heavy chain variable region (VH) comprising VH CDRs 1, 2, 3 and a light chain variable region (VL) comprising VL CDRs 1, 2, 3, wherein the VH CDRs 1, 2, 3 and the VL CDRs 1, 2, 3 are identical to the VH CDRs 1, 2, 3 and the VL CDRs 1, 2, 3 of the scFvs shown in FIG. 21.

[0307] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain (VH) containing one, two, or three of VH CDR1 with zero, one or two amino acid insertions, deletions, or substitutions; VH CDR2 with zero, one or two amino acid insertions, deletions, or substitutions; VH CDR3 with zero, one or two amino acid insertions, deletions, or substitutions; and a light chain variable domain (VL) containing one, two, or three of VL CDR1 with zero, one or two amino acid insertions, deletions, or substitutions; VL CDR2 with zero, one or two amino acid insertions, deletions, or substitutions; VL CDR3 with zero, one or two amino acid insertions, deletions, or Attorney Docket No.: 60846-0002W01 substitutions; wherein the VH CDR1, VH CDR2, and VH CDR3 and the VL CDR1, VL CDR2, and VL CDR3 are selected from the CDRs in FIG. 19 and FIG. 20. The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence. In some embodiments, the CDR is determined based on Chothia numbering scheme. In some embodiments, the CDR is determined based on Kabat numbering scheme. In some embodiments, the CDR is determined based on IMGT numbering scheme. In some embodiments, the CDR is determined based on contact definition. In some embodiments, the CDR is determined based on a combination of numbering schemes. Details of different numbering schemes can be found, e.g., in Dondelinger, M., et al. "Understanding the significance and implications of antibody numbering and antigen-binding surface / residue definition." Frontiers in Immunology 9 (2018): 2278, which is incorporated herein by reference in its entirety.

[0308] The disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in FIG. 19 and FIG. 20, or have sequences as shown in FIG. 21. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptides bind to CD3 or DLL3.

[0309] Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to CD3 will retain an ability to bind to CD3; and a fragment of an antibody that binds to DLL3 will retain an ability to bind to DLL3. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site. Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv Attorney Docket No.: 60846-0002W01 polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0310] The present disclosure provides various antibodies and antigen-binding fragments thereof derived from anti-CD3 or anti-DLL3 antibodies described herein. In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting examples of antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgEl, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, VH) and multiple constant domains (or constant regions), bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, VL) and one constant domain (or constant region), each bind to one heavy chain via disulfide binding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR).

[0311] These hypervariable regions, known as the complementary determining regions (CDRs), form loops that comprise the antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting the beta-sheet structure, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.

[0312] Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Attorney Docket No.: 60846-0002W01

[0313] Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, T.T. and Kabat, E.A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68(l-3):9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94 (Jan .1998); Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety.

[0314] The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.

[0315] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgGl, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions." Frontiers in Immunology 5 (2014); Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular Immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0316] The antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, camelid). Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide. The term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Attorney Docket No.: 60846-0002W01

[0317] Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a singlechain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., the heavy chain and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.

[0318] Fragments of antibodies are suitable for use in the methods described herein are also provided. The Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain. F(ab')2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0319] Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0320] Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0321] Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.

[0322] Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgGi molecules) spontaneously form protein aggregates containing antibody homodimers and other higher- order antibody multi mers.

[0323] Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4-(mal eimidomethyl)cy cl ohexane-1 -carboxylate) and SATA (N-succinimidyl S-acethylthio-acetate) can be used to form antibody multimers. An Attorney Docket No.: 60846-0002W01 exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Set. U.S.A. 94: 7509-7514, 1997). Antibody homodimers can be converted to Fab’2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0324] In some embodiments, the multi-specific antibody is a bi-specific antibody. Bispecific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96 / 27011, which is incorporated by reference in its entirety.

[0325] Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0326] Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution). Non -limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin). The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human). Attorney Docket No.: 60846-0002W01

[0327] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthracin, maytansinoids such as DM-1 and DM-4, di one, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs).

[0328] In some embodiments, the antigen binding fragment can form a part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane- and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 4 IBB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.

[0329] In some embodiments, the scFv has one heavy chain variable domain, and one light chain variable domain. In some embodiments, the scFv has two heavy chain variable domains, and two light chain variable domains.

[0330] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of illustration, the Attorney Docket No.: 60846-0002W01 comparison of sequences and determination of percent identity between two sequences can be accomplished, e.g., using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0331] The antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific), human antibodies, chimeric antibodies (e.g., human-camelid chimera), single-chain antibodies, intracellularly- made antibodies (i.e., intrabodies), and antigen-binding fragments thereof.

[0332] In some embodiments, the antibodies or antigen-binding fragments thereof comprises an Fc region (or Fc domain) that can be originated from various types (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. In some embodiments, the Fc region is originated from an IgG antibody or antigen-binding fragment thereof. In some embodiments, the Fc region comprises one, two, three, four, or more heavy chain constant regions. In some embodiments, the Fc region comprises one, two, or more heavy chain hinge regions.

[0333] The present disclosure also provides an antibody or antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment as described herein. The cross-competing assay is known in the art, and is described e.g., in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gpl20 exterior envelope glycoprotein." Journal of Virology 70.3 (1996): 1863-1872, which is incorporated herein reference in its entirety. In one aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment as described herein. The epitope binning assay is known in the art, and is described e.g., in Estep et al. "High throughput solution-based measurement of antibody-antigen affinity and epitope binning." MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, which is incorporated herein reference in its entirety.

[0334] In some embodiments, the antibodies or antigen binding fragments are humanized antibodies. Humanization percentage means the percentage identity of the heavy chain or light chain variable region sequence as compared to human antibody sequences in International Immunogenetics Information System (IMGT) database. The top hit means that the heavy chain or light chain variable region sequence is closer to a particular species than to Attorney Docket No.: 60846-0002W01 other species. For example, top hit to human means that the sequence is closer to human than to other species. Top hit to human and Macaca fascicularis means that the sequence has the same percentage identity to the human sequence and the Macaca fascicularis sequence, and these percentages identities are highest as compared to the sequences of other species. In some embodiments, humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. A detailed description regarding how to determine humanization percentage and how to determine top hits is known in the art, and is described, e.g., in Jones, et al. "The INNs and outs of antibody nonproprietary names." MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. A high humanization percentage often has various advantages, e.g., more safe and more effective in humans, more likely to be tolerated by a human subject, and / or less likely to have side effects.

[0335] PROTEIN COMPLEXES

[0336] Provided herein are protein complexes including (a) a fragment crystallizable (Fc) region; (b) a first antigen -binding moiety that specifically binds to a first cancer antigen (e.g., any of the cancer antigens described herein); and (c) a second antigen-binding moiety that specifically binds to a first effector cell antigen (e.g., any of the effector cell antigens described herein). In some embodiments, the second antigen-binding moiety is linked to the Fc region via a first linker peptide (e.g., any of the protease cleavable linkers described herein). In some embodiments, at least one therapeutic agent (e.g., any of the therapeutic agents described herein) is conjugated to the Fc region. In some embodiments, the first linker peptide is cleavable by a protease (e.g., any of the proteases described herein) within tumor microenvironment. In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety form a T cell engager (TCE) when the first linker peptide is cleaved. In some embodiments, the Fc region can be any of the silenced Fc regions described herein.

[0337] In some embodiments, the protein complexes further include: a third antigen-binding moiety that specifically binds to a second cancer antigen (e.g., any of the cancer antigens described herein), and a fourth antigen-binding moiety that specifically binds to a second effector cell antigen (e.g., any of the effector cell antigens described herein). The first and second effector cell antigens may be the same or different. In some embodiments, the fourth antigen-binding moiety is linked to the Fc region via a second linker peptide (e.g., any of the Attorney Docket No.: 60846-0002W01 protease cleavable linkers described herein). In some embodiments, the second linker peptide is cleavable by a protease (e.g., any of the proteases described herein) within tumor microenvironment. In some embodiments, the third antigen-binding moiety and the fourth antigen-binding moiety form a T cell engager (TCE) when the second linker peptide is cleaved.

[0338] An exemplary structure of the protein complexes described herein is shown in FIG. 9A as “P2-ADC.” Additional exemplary structures are provided in FIG. 14.

[0339] In some embodiments, the first antigen-binding moiety includes a first VHH, the second antigen-binding moiety includes a second VHH, the third antigen-binding moiety includes a third VHH, and the fourth antigen-binding moiety includes a fourth VHH. The first, second, third, and / or fourth VHHs may be the same or different. In some embodiments, the first and third VHHs are the same. In some embodiments, the second and fourth VHHs are the same. In some embodiments, at least one (e.g., 1 or 2) of the first and third VHHs are selected from any of the anti-DLL3 VHHs described herein. In some embodiments, at least one (e.g., 1 or 2) of the second and fourth VHHs are selected from any of the anti-CD3 VHHs described herein.

[0340] In some embodiments, the first antigen -binding moiety includes a first Fab, the second antigen-binding moiety includes a second Fab, the third antigen-binding moiety includes a third Fab, and the fourth antigen-binding moiety includes a fourth Fab. The first, second, third, and / or fourth Fabs may be the same or different. In some embodiments, the first and third Fabs are the same. In some embodiments, the second and fourth Fabs are the same. In some embodiments, at least one (e.g., 1 or 2) of the first and third Fabs are derived from any of the anti-DLL3 scFvs described herein. In some embodiments, at least one (e.g., 1 or 2) of the second and fourth Fabs are derived from any of the anti-CD3 scFvs described herein.

[0341] In some embodiments, the first antigen-binding moiety includes a first scFv, the second antigen-binding moiety includes a second scFv, the third antigen-binding moiety includes a third scFv, and the fourth antigen-binding moiety includes a fourth scFv. The first, second, third, and / or fourth scFvs may be the same or different. In some embodiments, the first and third scFvs are the same. In some embodiments, the second and fourth scFvs are the same. In some embodiments, at least one (e.g., 1 or 2) of the first and third scFvs are selected from any of the anti-DLL3 scFvs described herein. In some embodiments, at least one (e.g., 1 Attorney Docket No.: 60846-0002W01 or 2) of the second and fourth scFvs are selected from any of the anti-CD3 scFvs described herein.

[0342] In some embodiments, the first antigen -binding moiety includes a first Fab, the second antigen-binding moiety includes a first scFv, the third antigen-binding moiety includes a second Fab, and the fourth antigen-binding moiety includes a second scFv. The first and second Fabs may be the same or different. The first and second scFvs may be the same or different. In some embodiments, the first and second Fabs are the same. In some embodiments, the first and second scFvs are the same. In some embodiments, at least one (e.g., 1 or 2) of the first and second Fabs are derived from any of the anti-DLL3 scFvs described herein. In some embodiments, at least one (e.g., 1 or 2) of the first and second scFvs are selected from any of the anti-CD3 scFvs described herein.

[0343] In some embodiments, the first antigen-binding moiety includes a first VHH, the second antigen-binding moiety includes a first Fab, the third antigen-binding moiety includes a second VHH, and the fourth antigen-binding moiety includes a second Fab. The first and second VHHs may be the same or different. The first and second Fabs may be the same or different. In some embodiments, the first and second VHHs are the same. In some embodiments, the first and second Fabs are the same. In some embodiments, at least one (e.g., 1 or 2) of the first and second VHHs are selected from any of the anti-DLL3 VHHs described herein. In some embodiments, at least one (e.g., 1 or 2) of the first and second Fabs are derived from any of the anti-CD3 scFvs described herein.

[0344] In some embodiments, the first antigen-binding moiety includes a first VHH, the second antigen-binding moiety includes a first scFv, the third antigen-binding moiety includes a second VHH, and the fourth antigen-binding moiety includes a second scFv. The first and second VHHs may be the same or different. The first and second scFvs may be the same or different. In some embodiments, the first and second VHHs are the same. In some embodiments, the first and second scFvs are the same. In some embodiments, at least one (e.g., 1 or 2) of the first and second VHHs are selected from any of the anti-DLL3 VHHs described herein. In some embodiments, at least one (e.g., 1 or 2) of the first and second scFvs are derived from any of the anti-CD3 scFvs described herein.

[0345] In some embodiments, the first antigen-binding moiety includes a first scFv, the second antigen-binding moiety includes a first Fab, the third antigen-binding moiety includes a second scFv, and the fourth antigen-binding moiety includes a second Fab. The first and Attorney Docket No.: 60846-0002W01 second scFvs may be the same or different. The first and second Fabs may be the same or different. In some embodiments, the first and second scFvs are the same. In some embodiments, the first and second Fabs are the same. In some embodiments, at least one (e.g., 1 or 2) of the first and second scFvs are selected from any of the anti-DLL3 scFvs described herein. In some embodiments, at least one (e.g., 1 or 2) of the first and second Fabs are derived from any of the anti-CD3 scFvs described herein.

[0346] In some embodiments, the first antigen -binding moiety includes a first Fab, the second antigen-binding moiety includes a first VHH, the third antigen-binding moiety includes a second Fab, and the fourth antigen-binding moiety includes a second VHH. The first and second Fabs may be the same or different. The first and second VHHs may be the same or different. In some embodiments, the first and second Fabs are the same. In some embodiments, the first and second VHHs are the same. In some embodiments, at least one (e.g., 1 or 2) of the first and second Fabs are derived from any of the anti-DLL3 scFvs described herein. In some embodiments, at least one (e.g., 1 or 2) of the first and second VHHs are selected from any of the anti-CD3 VHHs described herein.

[0347] In some embodiments, the first antigen-binding moiety includes a first scFv, the second antigen-binding moiety includes a first VHH, the third antigen-binding moiety includes a second scFv, and the fourth antigen-binding moiety includes a second VHH. The first and second scFvs may be the same or different. The first and second VHHs may be the same or different. In some embodiments, the first and second scFvs are the same. In some embodiments, the first and second VHHs are the same. In some embodiments, at least one (e.g., 1 or 2) of the first and second scFvs are selected from any of the anti-DLL3 scFvs described herein. In some embodiments, at least one (e.g., 1 or 2) of the first and second VHHs are selected from any of the anti-CD3 VHHs described herein.

[0348] In some embodiments, the protein complexes described herein includes a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 586, 588, 590, 592, 594, 596 and 598; and the second polypeptide includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 587, 589, 591, 593, 595, 597 and 599. Attorney Docket No.: 60846-0002W01

[0349] In some embodiments, the protein complexes described herein further include: a fifth antigen-binding moiety that specifically binds to a third cancer antigen (any of the cancer antigens described herein), and a sixth antigen-binding moiety that specifically binds to a fourth cancer antigen (any of the cancer antigens described herein). In some embodiments, the fifth antigen-binding moiety is linked to the first antigen-binding moiety. In some embodiments, the sixth antigen-binding moiety is linked to the third antigen-binding moiety.

[0350] In some embodiments, first antigen-binding moiety includes a first VHH, the second antigen-binding moiety includes a first scFv, the third antigen-binding moiety includes a second VHH, the fourth antigen-binding moiety includes a second scFv, the fifth antigenbinding moiety includes a third VHH, and the sixth antigen-binding moiety includes a fourth VHH. The first, second, third, and / or fourth VHHs may be the same or different. The first and second scFvs may be the same or different. In some embodiments, at least one (e.g., 1, 2, 3, or 4) of the first, second, third, and fourth VHHs are selected from any of the anti-DLL3 VHHs described herein. In some embodiments, at least one (e.g., 1 or 2) of the first and second scFvs are selected from any of the anti-CD3 scFvs described herein. An exemplary structure is shown in FIG. 9A as “Pl -ADC.”

[0351] In some embodiments, the protein complexes described herein includes a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 579-585; and the second polypeptide includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 579-585.

[0352] In some embodiments, the protein complexes described herein further include: a third antigen-binding moiety that specifically binds to a second cancer antigen (e.g., any of the cancer antigens described herein). In some embodiments, the third anti gen -binding moiety is linked to the Fc region. In some embodiments, the third antigen-binding moiety and the Fc region form an antibody-drug conjugate (ADC) when the first linker peptide is cleaved. An exemplary structure of the protein complexes described herein is shown in FIG. 9A as “P3- ADC.”

[0353] In some embodiments, the first antigen-binding moiety includes a VHH, the second antigen-binding moiety includes a first scFv, and the third antigen-binding moiety includes a second scFv. In some embodiments, the VHH is selected from any of the anti-DLL3 VHHs Attorney Docket No.: 60846-0002W01 described herein. In some embodiments, the first scFv is selected from any of the anti-CD3 scFvs described herein. In some embodiments, the second scFv is selected from any of the anti-DLL3 scFvs described herein.

[0354] In some embodiments, the protein complexes described herein includes a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 600, 602, 604, 606, 608, and 610; and the second polypeptide includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 601, 603, 604, 607, 609, and 611.

[0355] In some embodiments, the protein complexes described herein further include: a fourth antigen-binding moiety that specifically binds to a third cancer antigen (e.g., any of the cancer antigens described herein). In some embodiments, the fourth antigen-binding moiety is linked to the first antigen-binding moiety. In some embodiments, the third antigen-binding moiety and the Fc region form an antibody-drug conjugate (ADC) when the first linker peptide is cleaved. In some embodiments, the fourth antigen-binding moiety, the first antigen-binding moiety, and the second antigen-binding moiety form a TCE when the first linker peptide is cleaved. An exemplary structure of the protein complexes described herein is shown in FIG. 9B as “P4-ADC.”

[0356] In some embodiments, the first antigen-binding moiety includes a first VHH, the second antigen-binding moiety includes a first scFv, the third antigen-binding moiety includes a second scFv, and the fourth antigen-binding moiety includes a second VHH. The first and second VHHs may be the same or different. In some embodiments, at least one (e.g., 1 or 2) of the first and second VHHs are selected from any of the anti-DLL3 VHHs described herein. In some embodiments, the first scFv is selected from any of the anti-CD3 scFvs described herein. In some embodiments, the second scFv is selected from any of the anti- DLL3 scFvs described herein.

[0357] In some embodiments, the protein complexes described herein includes a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 612, 614, 616, 618, 620, and 622; and the second polypeptide includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, Attorney Docket No.: 60846-0002W01

[0358] 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 613, 614, 617, 619, 621, and 623.

[0359] In some embodiments, the protein complexes described herein further include: a fourth antigen-binding moiety that specifically binds to a third cancer antigen (e.g., any of the cancer antigens described herein). In some embodiments, the fourth antigen-binding moiety is linked to the third antigen-binding moiety. In some embodiments, the fourth antigenbinding moiety, the third antigen-binding moiety and the Fc region form an antibody-drug conjugate (ADC) when the first linker peptide is cleaved. In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety form a TCE when the first linker peptide is cleaved. An exemplary structure of the protein complexes described herein is shown in FIG. 9B as “P5-ADC.”

[0360] In some embodiments, the first antigen-binding moiety includes a first scFv, the second antigen-binding moiety includes a second scFv, the third antigen-binding moiety includes a first VHH, and the fourth antigen-binding moiety includes a second VHH. The first and second VHHs may be the same or different. In some embodiments, the first scFv is selected from any of the anti-DLL3 scFvs described herein. In some embodiments, the second scFv is selected from any of the anti-CD3 scFvs described herein.

[0361] In some embodiments, the protein complexes described herein includes a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 624, 626, 628, 630, 632, and 634; and the second polypeptide includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 625, 627, 629, 631, 633, and 635.

[0362] After cleavage of the first and / or second linker peptides, the TCE described herein may include one or more (e.g., 1, 2, 3, 4 or 5) cancer antigen-binding moi eties and one or more (e.g., 1, 2, 3, 4, or 5) effector cell antigen-binding moieties. Each protein complex, after cleavage, may generate 1, 2, 3, 4, or 5 TCE molecules and / or 1, 2, 3, 4, or 5 ADC molecules.

[0363] ANTIBODY AND PROTEIN COMPLEX CHARACTERISTICS

[0364] In some embodiments, the antibodies, antigen-binding fragments thereof, or protein complexes thereof described herein can bind to DLL3 (e.g., human or monkey DLL3). In Attorney Docket No.: 60846-0002W01 some embodiments, the monkey DLL3 is cynomolgus macaque DLL3. In some embodiments, the antibodies, antigen-binding fragments thereof, or protein complexes thereof described herein can bind to both human DLL3 and non-human DLL3 (e.g., monkey DLL3).

[0365] In some embodiments, the antibodies, antigen-binding fragments thereof, or protein complexes thereof described herein have a DLL3-binding capability (e.g., determined by BLI) that is at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, at least or about 200% as compared to that of a benchmark molecule (e.g., AMG-757 analog or INN analog) or a control molecule (e.g., an isotype control).

[0366] In some embodiments, the antibodies, antigen-binding fragments thereof, or protein complexes thereof described herein have a binding ability of DLL3 -expressing cells (e.g., SHP-77 cells; as determined by flow cytometry) that is at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, at least or about 200% as compared to that of a benchmark molecule (e.g., AMG-757 analog or INN analog) or a control molecule (e.g., an isotype control).

[0367] In some embodiments, the antibodies, antigen-binding fragments thereof, or protein complexes thereof described herein can specifically bind to DLL3 (e.g., determined by BLI) with a dissociation rate (Koff) of less than 0.1 s’1, less than 0.01 s’1, less than 0.001 s’1, less than 0.0009 s’1, less than 0.0007 s’1, less than 0.0005 s’1, less than 0.0003 s’1, less than 0.0001 s’1, or less than 0.00001 s’1. In some embodiments, the dissociation rate (Koff) is greater than 0.1 s’1, greater than 0.01 s’1, greater than 0.001 s’1, greater than 0.0009 s’1, greater than 0.0007 s’1, greater than 0.0005 s’1, greater than 0.0003 s’1, greater than 0.0001 s’1, or greater than 0.00001 s’1. In some embodiments, kinetic association rates (Kon) is greater than 1 x 102 / Ms, greater than 1 x 103 / Ms, greater than 1 x 104 / Ms, greater than 1 x 105 / Ms, greater than 2 x 105 / MS, greater than 3 x 105 / Ms, greater than 4 x 105 / Ms, greater than 5 x 105 / Ms, greater than 6 x 105 / Ms, or greater than 7 x 105 / Ms. In some embodiments, kinetic association rates (Kon) is less than 1 x 102 / Ms, less than 1 x 103 / Ms, less than 1 x 104 / Ms, less than 1 x Attorney Docket No.: 60846-0002W01

[0368] 105 / MS, less than 2 x 105 / Ms, less than 3 x 105 / Ms, less than 4 x 105 / Ms, less than 5 x 105 / MS, less than 6 x 105 / Ms, or less than 7 x 105 / Ms.

[0369] Affinities can be deduced from the quotient of the kinetic rate constants (KD = Koff / Kon). In some embodiments, KD is less than 1 x 10'4M, less than 1 x 10'5M, less than 1 x 10'6M, less than 1 x 10'7M, less than 1 x 10'8M, less than 1 x 10'9M, or less than 1 x 1O'10M. In some embodiments, the KD is less than 100 nM, 80 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10'4M, greater than 1 x 10'5M, greater than 1 x 10'6M, greater than 1 x 10'7M, greater than 1 x 10'8M, greater than 1 x 10'9M, greater than 1 x 1O'10M, greater than 1 x 10'11M, or greater than 1 x 10'12M. Furthermore, Ka can be deduced from KD by the formula Ka=l / Ko.

[0370] General techniques for measuring the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the affinity of an antibody for an antigen is determined by Bio-Layer Interferometry (BLI) described herein.

[0371] In some embodiments, the protein complexes described herein having a first and / or second cleavable linker peptides (e.g., any of the protease cleavable linkers described herein), when treated with a protease (e.g., any of the proteases described herein), generates fragments thereof with a cleavage rate that is comparable to a control molecule (e.g., a commercial masked TCE molecule having a cleavable linker peptide).

[0372] In some embodiments, the antibodies, antigen-binding fragments thereof, or protein complexes thereof described herein have a binding ability of cells not expressing DLL3 (e.g., 293T cells; as determined by flow cytometry) that is less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 100%, less than 120%, less than 150%, less than 200%, less than 300%, or less than 500% as compared to that of a benchmark molecule (e.g., AMG-757 analog). In some embodiments, the antibodies, antigen-binding fragments thereof, or protein complexes thereof described herein have a binding ability of DLL3 -expressing cells (e.g., 293T-DLL3 cells; as determined by flow cytometry) that is at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, at least or about 200% as compared to that of a benchmark molecule (e.g., AMG-757 analog). Attorney Docket No.: 60846-0002W01

[0373] In some embodiments, the protein complexes described herein, e.g., any of the protein complexes described herein that can be cleaved by a protease (e.g., any proteases described herein) to form one or more TCE molecules, have a binding ability to cells expressing an effector cell antigen (e.g., Jurkat cells; as determined by flow cytometry) that is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% as compared to that of a benchmark molecule (e.g., AMG-757 analog). As a result, the protein complexes described herein can effectively mask the TCE molecule before cleavage.

[0374] In some embodiments, the protein complexes described herein, e.g., any of the protein complexes described herein that can be cleaved by a protease (e.g., any proteases described herein) to form one or more TCE molecules, have a T cell activation strength (e.g., as determined by flow cytometry using one or more T cell activation markers) that is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% as compared to that of the one or more TCE molecules that are cleaved from the protein complexes.

[0375] In some embodiments, the ADCs described herein (e.g., any of the protein complexes described herein conjugated with at least one therapeutic agent), when combined with effector cells (e.g., PBMCs), have a target cell killing efficacy that is 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%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold as compared to that of the ADCs alone, or that of a combination of the corresponding protein complexes and the effector cells.

[0376] In some embodiments, the ADCs described herein (e.g., any of the protein complexes described herein conjugated with at least one therapeutic agent), when combined with effector cells (e.g., PBMCs or T cells), do not induce killing of the effector cells.

[0377] In some embodiments, the antibodies, antigen-binding fragments thereof, or protein complexes thereof described herein have a tumor growth inhibition percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibodies, Attorney Docket No.: 60846-0002W01 antigen-binding fragments thereof, or protein constructs thereof described herein have a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:

[0378] TGI (%) = [l-(Ti-T0) / (Vi-V0)]xl00

[0379] Ti is the average tumor volume in the treatment group on day i. TO is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

[0380] In some embodiments, the antibodies, antigen-binding fragments thereof, or protein complexes thereof described herein have the antibody-dependent cell-mediated cytotoxicity (ADCC) or the complement dependent cytotoxicity (CDC) that is less than or about 50%, less than or about 60%, less than or about 70%, less than or about 80%, less than or about 90%, less than or about 100%, less than or about 110%, less than or about 120%, less than or about 130%, less than or about 140%, less than or about 150%, less than or about 200% as compared to that of an isotype antibody control. In some embodiments, the antibodies, antigen-binding fragments thereof, or protein constructs thereof described herein can decrease antibody-dependent cell-mediated cytotoxicity (ADCC) or the complement dependent cytotoxicity (CDC) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, 20 folds, or 100 folds, as compared to that of an isotype antibody control.

[0381] RECOMBINANT POLYPEPTIDES

[0382] Provided herein are, inter alia, recombinant polypeptides having the functionality of a T cell engager as well as an antibody-drug conjugate. The recombinant polypeptides provided herein including embodiments thereof include a (i) T cell engager portion, which includes a cancer antigen binding domain and an effector cell binding domain, and (ii) an antibody-drug conjugate portion, which includes an Fc domain attached a plurality of therapeutic moieties. The T cell engager portion and the antibody-drug conjugate portion of the recombinant polypeptides provided herein including embodiments thereof are connected through protease Attorney Docket No.: 60846-0002W01 cleavable linker. Absent cleavage of the protease cleavable linker the effector cell binding domain remains structurally occluded and not accessible for binding its cognate receptor. In its uncleaved form the recombinant polypeptide may bind to a cancer cell through the cancer antigen binding domain and enter the cancer cell by receptor-mediated endocytosis thereby releasing the plurality of therapeutic moi eties inducing cancer-specific cytotoxicity.

[0383] Upon cleavage of the protease cleavable linker, for example, by a cancer / tumor specific protease, the T cell engager portion and the antibody-drug conjugate portion are separated and the effector cell binding domain becomes accessible. The cancer antigen binding domain, which is still connected to the effector cell binding domain binds to a cancer antigen while the effector cell binding domain binds to an effector cell thereby sequestering the cancer cell and the effector cell into close proximity to provide for efficient targeted cell killing of the cancer cell through the effector cell.

[0384] Furthermore, the Fc dimerizing domain included in the recombinant polypeptide provided herein including embodiments thereof may form part of an Fc domain by dimerizing with an Fc dimerizing domain of another recombinant polypeptide provided herein including embodiments thereof. Said Fc domain may not detectably bind to its cognate receptor (e.g., FcyRIIIa) and therefore detectably activating effector cells (e.g., T cells, Nk cells). Therefore, the recombinant polypeptides provided herein including embodiments thereof may not induce an antibody-dependent cellular cytotoxicity (ADCC) mediated through their Fc domain. An Fc domain that does not bind to its cognate receptor at a detectable level relative to a standard control, may also be referred to herein as a “silenced Fc” or “silenced Fc domain.” The recombinant proteins as provided herein including embodiments thereof may induce only limited cytokine release. The recombinant polypeptides provided herein including embodiments thereof are surprisingly effective compared to traditional bispecific antibodies as they exhibit high specificity and effectivity while lacking undesirable adverse side effects.

[0385] Thus, in an aspect is provided, a recombinant polypeptide including: (i) a cancer antigen binding domain bound to an effector cell binding domain; (ii) an Fc dimerizing domain covalently bound to at least one therapeutic moiety; and (iii) a protease cleavable linker connecting the cancer antigen binding domain with the Fc dimerizing domain through binding the effector cell binding domain to the Fc dimerizing domain. Attorney Docket No.: 60846-0002W01

[0386] A “cancer antigen binding domain” as provided herein refers to a peptide domain capable of selectively binding to a cancer antigen. A cancer antigen binding domain may covalently or non-covalently bind to a cancer antigen. Non-limiting examples of cancer antigen binding domains include single chain antibodies, antibody variants or fragments thereof, antibodies or fragments thereof. In embodiments, the cancer antigen binding domain is a Fab. In embodiments, the cancer antigen binding domain is a single domain antibody (sdAb).

[0387] An “effector cell binding domain” as provided herein refers to a peptide domain capable of selectively binding to an effector cell ligand expressed on an effector cell. The term “effector cell ligand” as provided herein refers to a cell surface molecule expressed on an effector cell of the immune system (e.g., a cytotoxic T cell, a helper T cell, a B cell, a natural killer cell). Upon binding of the effector cell binding domain to the effector cell ligand expressed on the effector cell, the effector cell is activated and able to exert its function (e.g., selective killing or eradication of malignant, infected or otherwise unhealthy cells). In embodiments, the effector cell ligand is a CD3 protein. In embodiments, the effector cell ligand is a CD 16 protein. In embodiments, the effector cell ligand is a CD32 protein. In embodiments, the effector cell ligand is a NKp46 protein. Examples of effector cells include without limitation, cytotoxic T cells, helper T cells, B cells, and natural killer cells. Nonlimiting examples of effector cell binding domains include single chain antibodies, antibody variants or fragments thereof, antibodies or fragments thereof. In embodiments, the effector cell binding domain is a Fab. In embodiments, the effector cell binding domain is a single domain antibody (sdAb).

[0388] In embodiments, the cancer antigen binding domain is bound to the effector cell binding domain through a chemical linker. In embodiments, the chemical linker is a bond.

[0389] In embodiments, the cancer antigen binding domain is an antibody domain. An “antibody domain” as provided herein refers to a polypeptide capable of binding to an antigen and including one or more structural domains of an antibody or fragment thereof. Thus, an antibody domain may be an antibody or functional fragment thereof or an antibody variant or functional fragment thereof. In embodiments, the cancer antigen binding domain is an scFv, a Fab, a single domain antibody or a bispecific antibody. In embodiments, the cancer antigen binding domain is an scFv. In embodiments, the cancer antigen binding domain is a Fab. In Attorney Docket No.: 60846-0002W01 embodiments, the cancer antigen binding domain is a single domain antibody. In embodiments, the cancer antigen binding domain is a bispecific antibody.

[0390] In embodiments, the single domain antibody is a VHH. In embodiments, the single domain antibody is a llama VHH, a camelid VHH, or a shark VHH. In embodiments, the single domain antibody is a llama VHH. In embodiments, the single domain antibody is a camelid VHH. In embodiments, the single domain antibody is a shark VHH.

[0391] In embodiments, the cancer antigen binding domain is Trastuzumab (Herceptin®), Margetuximab-cmkb, Pertuzumab, Ramucirumab, Cetuximab, Panitumumab, Obinutuzumab, Rituximab, Ofatumumab, Alemtuzumab, Necitumumab, Mogamulizumab, Tafasitamab, Daratumumab, Isatuximab, Elotuzumab, Bevacizumab, Naxitamab-gqgk, Dinutuximab, Olaratumumab, Denosumab, Enfortumab vedotin, Trastuzumab deruxtecan, Trastuzumab emtansine, Sacituzumab govitecan-hziy, Tisotumab vedotin, Inotuzumab ozogamicin, Moxetumomab pasudotox, Gemtuzumab ozogamicin, Loncastuximab tesirine, Ibritumomab tiuxetan, Brentuximab vedotin, Polatuzumab vedotin, Mirvetuximab soravtansine, Amivantamab, Blinatumomab, Epcoritama, Glofitamab, Mosunetuzumab, Tebentafusp-tebn or Teclistimab.

[0392] The recombinant polypeptides provided herein are, inter alia, useful therapeutic agents, because of their ability to bind cancer antigens effectively and with high specificity. In embodiments, the cancer antigen is an adult acute lymphoblastic leukemia (ALL) antigen, chronic lymphocytic leukemia (CLL) antigen, non-Hodgkin lymphoma (NHL) antigen, adult acute myeloid leukemia (AML) antigen, multiple myeloma (MML) antigen, leukemia antigen, lymphoma antigen, urothelial carcinoma antigen, colorectal cancer antigen, nonsmall cell lung carcinoma (NSCLC) antigen, lung cancer antigen, glioblastoma antigen, gastric cancer antigen, hepatocellular carcinoma antigen, metastatic melanoma antigen, endometrial cancer antigen, secondary bone cancer antigen, neuroblastoma antigen, soft tissue carcinoma antigen, breast cancer antigen, cervical cancer antigen, bladder cancer antigen, nasopharyngeal cancer antigen, or ovarian cancer antigen. In embodiments, the cancer antigen is an adult acute lymphoblastic leukemia (ALL) antigen. In embodiments, the cancer antigen is a chronic lymphocytic leukemia (CLL) antigen. In embodiments, the cancer antigen is a non-Hodgkin lymphoma (NHL) antigen. In embodiments, the cancer antigen is an adult acute myeloid leukemia (AML) antigen. In embodiments, the cancer antigen is a multiple myeloma (MML) antigen. In embodiments, the cancer antigen is a leukemia antigen. Attorney Docket No.: 60846-0002W01

[0393] In embodiments, the cancer antigen is a lymphoma antigen. In embodiments, the cancer antigen is an urothelial carcinoma antigen. In embodiments, the cancer antigen is a colorectal cancer antigen. In embodiments, the cancer antigen is a non-small cell lung carcinoma (NSCLC) antigen. In embodiments, the cancer antigen is a lung cancer antigen. In embodiments, the cancer antigen is a glioblastoma antigen. In embodiments, the cancer antigen is a gastric cancer antigen. In embodiments, the cancer antigen is a hepatocellular carcinoma antigen. In embodiments, the cancer antigen is a metastatic melanoma antigen. In embodiments, the cancer antigen is an endometrial cancer antigen. In embodiments, the cancer antigen is a secondary bone cancer antigen. In embodiments, the cancer antigen is a neuroblastoma antigen. In embodiments, the cancer antigen is a soft tissue carcinoma antigen. In embodiments, the cancer antigen is a breast cancer antigen. In embodiments, the cancer antigen is a cervical cancer antigen. In embodiments, the cancer antigen is a bladder cancer antigen. In embodiments, the cancer antigen is a nasopharyngeal cancer antigen. In embodiments, the cancer antigen is an ovarian cancer antigen.

[0394] In embodiments, the cancer antigen is CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD96, CD123, CDH17, CEA, CLDN6, CLDN18.2, cMET, CCR4DLL3, DLL4, DKK, NECTIN4, Claudin, LIVE6, BCMA, HER2, HER3, HLA- G, KLK2, LGR5, MAGEA4 / 8, mesothelin, angiopoietin, FGF, FGFR, MUC16, PRAME, PSMA, STEAP1, TMEFF2, PD-L1, EGFR, EGFRvIII, ENPP3, EpCAM, gplOO, GPC3, GUCY2C, VEGF, VEGFR2, CTLA-4, PD-1, RANK-L, GD2, SLAMF7, PDGFRA, TROP-2, FR-alpha, FcRH5, FLT3, GPRC5D, ROR1, 5T4, B7-H3, or B7-H4. In embodiments, the cancer antigen is CD3. In embodiments, the cancer antigen is CD 19. In embodiments, the cancer antigen is CD20. In embodiments, the cancer antigen is CD22. In embodiments, the cancer antigen is CD25. In embodiments, the cancer antigen is CD30. In embodiments, the cancer antigen is CD33. In embodiments, the cancer antigen is CD37. In embodiments, the cancer antigen is CD38. In embodiments, the cancer antigen is CD52. In embodiments, the cancer antigen is CD56. In embodiments, the cancer antigen is CD96. In embodiments, the cancer antigen is CD123. In embodiments, the cancer antigen is CDH17. In embodiments, the cancer antigen is CEA. In embodiments, the cancer antigen is CLDN6. In embodiments, the cancer antigen is CLDN18.2. In embodiments, the cancer antigen is cMET. In embodiments, the cancer antigen is CCR4DLL3. In embodiments, the cancer antigen is DLL4. In embodiments, the cancer antigen is DKK. In embodiments, the cancer antigen is NECTIN4. Attorney Docket No.: 60846-0002W01

[0395] In embodiments, the cancer antigen is Claudin. In embodiments, the cancer antigen is LIVE6. In embodiments, the cancer antigen is BCMA. In embodiments, the cancer antigen is HER2. In embodiments, the cancer antigen is HER3. In embodiments, the cancer antigen is HLA-G. In embodiments, the cancer antigen is KLK2. In embodiments, the cancer antigen is LGR5. In embodiments, the cancer antigen is MAGEA4 / 8. In embodiments, the cancer antigen is mesothelin. In embodiments, the cancer antigen is angiopoietin. In embodiments, the cancer antigen is FGF. In embodiments, the cancer antigen is FGFR. In embodiments, the cancer antigen is MUC16. In embodiments, the cancer antigen is PRAME. In embodiments, the cancer antigen is PSMA. In embodiments, the cancer antigen is STEAP1. In embodiments, the cancer antigen is TMEFF2. In embodiments, the cancer antigen is PD-L1. In embodiments, the cancer antigen is EGFR. In embodiments, the cancer antigen is EGFRvIII. In embodiments, the cancer antigen is ENPP3. In embodiments, the cancer antigen is EpCAM. In embodiments, the cancer antigen is gplOO. In embodiments, the cancer antigen is, GPC3. In embodiments, the cancer antigen is GUCY2C. In embodiments, the cancer antigen is VEGF. In embodiments, the cancer antigen is VEGFR2. In embodiments, the cancer antigen is CTLA-4. In embodiments, the cancer antigen is PD-1. In embodiments, the cancer antigen is RANK-L. In embodiments, the cancer antigen is GD2. In embodiments, the cancer antigen is SLAMF7. In embodiments, the cancer antigen is PDGFRA. In embodiments, the cancer antigen is TROP-2. In embodiments, the cancer antigen is FR-alpha. In embodiments, the cancer antigen is FcRH5. In embodiments, the cancer antigen is FLT3. In embodiments, the cancer antigen is GPRC5D. In embodiments, the cancer antigen is ROR1. In embodiments, the cancer antigen is 5T4. In embodiments, the cancer antigen is B7- H3. In embodiments, the cancer antigen is B7-H4.

[0396] In embodiments, the effector cell binding domain is a CD3 binding domain, a CD28 binding domain, a PD-1 binding domain, a TIGIT binding domain, a Lag3 binding domain, a CD16A binding domain, a CD16B binding domain, a CD32A binding domain, a CD32B binding domain, a CD32C binding domain, a CD64 binding domain, or a SIRP-alpha binding domain. In embodiments, the effector cell binding domain is a CD3 binding domain. In embodiments, the effector cell binding domain is a CD28 binding domain. In embodiments, the effector cell binding domain is a PD-1 binding domain. In embodiments, the effector cell binding domain is a TIGIT binding domain. In embodiments, the effector cell binding domain is a Lag3 binding domain. In embodiments, the effector cell binding domain is a Attorney Docket No.: 60846-0002W01

[0397] CD16A binding domain. In embodiments, the effector cell binding domain is a CD16B binding domain. In embodiments, the effector cell binding domain is a CD32A binding domain. In embodiments, the effector cell binding domain is a CD32B binding domain. In embodiments, the effector cell binding domain is a CD32C binding domain. In embodiments, the effector cell binding domain is a CD64 binding domain. In embodiments, the effector cell binding domain is a SIRP-alpha binding domain.

[0398] In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 100 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 90 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 80 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 70 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 60 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 50 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 40 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 30 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 20 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 10 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 9 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 8 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 7 pM. In Attorney Docket No.: 60846-0002W01 embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 6 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 5 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 4 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 3 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 2 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 1 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 0.1 pM. In embodiments, the effector cell binding domain (i.e., first or second effector cell binding domain) binds to an effector cell ligand at an affinity of about 1 pM to about 0.01 pM.

[0399] In embodiments, the effector cell binding domain is a scFv or a VHH. In embodiments, the effector cell binding domain is a scFv (e.g., any of the anti-CD3 scFvs described herein). In embodiments, the effector cell binding domain is a VHH (e.g., any of the anti-CD3 VHHs described herein). In embodiments, the effector cell binding domain includes the sequence of SEQ ID NO: 1. In embodiments, the effector cell binding domain has the sequence of SEQ ID NO: 1. In some embodiments, the effector cell binding domain is an anti-CD3 antibody or antigen-binding fragment thereof (e.g., any of the anti-CD3 antibodies or antigen-binding fragments thereof described herein).

[0400] The recombinant polypeptides provided herein including embodiments thereof may include an Fc dimerizing domain or where two recombinant polypeptides are bound together, they may include an Fc domain or Fc region. An “Fc dimerizing domain” as referred to herein is a polypeptide including an antibody CH2 domain or fragment thereof bound (covalently and / or non-covalently) to an antibody CH3 domain or fragment thereof. Upon binding of two Fc dimerizing domains an antibody Fc region or Fc domain is formed. Thus, an Fc region or Fc domain may include a first Fc dimerizing domain non-covalently or covalently bound to a second Fc dimerizing domain. In embodiments, the CH3 domain of the Attorney Docket No.: 60846-0002W01 first Fc dimerizing domain is non-covalently bound to the CH3 domain of the second Fc dimerizing domain. In embodiments, the CH2 domain of the first Fc dimerizing domain is covalently bound to the CH2 domain of the second Fc dimerizing domain. In embodiments, the CH2 domain of the first Fc dimerizing domain is bound to the CH2 domain of the second Fc dimerizing domain through a disulfide linkage. In embodiments, the Fc dimerizing domain includes a CH2 domain and a CH3 domain. In embodiments, the Fc dimerizing domain includes from the N-terminus to the C-terminus a CH2 domain and a CH3 domain. In embodiments, the first Fc dimerizing domain includes from the N-terminus to the C-terminus a first CH2 domain and a first CH3 domain. In embodiments, the second Fc dimerizing domain includes from the N-terminus to the C-terminus a second CH2 domain and a second CH3 domain. Thus, in embodiments, the Fc dimerizing domain includes a CH2 domain and a CH3 domain.

[0401] The Fc domain or Fc region provided herein may include Fc dimerizing domains (i.e., a first Fc dimerizing domain and / or a second Fc dimerizing domain) with amino acid substitutions that render the domain inactive or silent relative to standard control (e.g., an Fc domain or Fc dimerizing domain not including said substitution). As described herein a silenced or inactive Fc domain, Fc dimerizing domain or Fc region does not detectably bind to its cognate receptor (e.g., FcR). Absent detectable binding of the Fc domain, Fc dimerzing domain or Fc region to its cognate receptor on an effector cell, the effector cell is not activated to exert its cytotoxic properties towards a cancer cell expressing said cognate receptor. Thus, in embodiments, the Fc dimerizing domain (i.e., a first Fc dimerizing domain and / or a second Fc dimerizing domain) does not induce antibody-dependent cellular cytotoxicity (ADCC) relative to a standard control.

[0402] In embodiments, the Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, a cysteine at a position corresponding to position 302, an alanine at a position corresponding to position 234, or an alanine at a position corresponding to position 235. In embodiments, the Fc dimerizing domain includes a glycine at a position corresponding to position 297. In embodiments, the Fc dimerizing domain includes a cysteine at a position corresponding to position 292. In embodiments, the Fc dimerizing domain includes a cysteine at a position corresponding to position 302. In embodiments, the Fc dimerizing domain includes an alanine at a position corresponding to position 234. In embodiments, the Fc dimerizing Attorney Docket No.: 60846-0002W01 domain includes an alanine at a position corresponding to position 235. In embodiments, the Fc dimerizing domain includes an amino acid substitution that decreases the Fc effector function of the Fc dimerizing domain. In embodiments, the amino acid substitution is at a position corresponding to position 234. In embodiments, the amino acid substitution is at a position corresponding to position 235. In embodiments, the Fc dimerizing domain includes the sequence of SEQ ID NO: 2. In embodiments, the Fc dimerizing domain includes the sequence of SEQ ID NO: 3. In embodiments, the Fc dimerizing domain is the sequence of SEQ ID NO: 2. In embodiments, the Fc dimerizing domain is the sequence of SEQ ID NO: 3.

[0403] The protease cleavable linker as provided herein is a cleavable peptide linker including a protease cleavage site. A "cleavage site" as used herein, refers to a recognizable site for cleavage of a portion of a linker described herein. Thus, a cleavage site may be found in the sequence of a protease cleavable linker as described herein, including embodiments thereof. In embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by a cleaving agent (e.g., a peptidyl sequence). Exemplary cleaving agents include proteins, enzymes, DNAzymes, RNAzymes, metals, acids, and bases. In embodiments, the protease cleavage site is a tumor-associated protease cleavage site. A "tumor-associated protease cleavage site" as provided herein is an amino acid sequence recognized by a protease, whose expression is specific for a tumor cell or tumor cell environment thereof. In embodiments, the protease cleavage site is a matrix metalloprotease (MMP) cleavage site, a disintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage site, a prostate specific antigen (PSA) protease cleavage site, a urokinase-type plasminogen activator (uPA) protease cleavage site, a membrane type serine protease 1 (MT-SP1) protease cleavage site or a legumain protease cleavage site. In embodiments, the matrix metalloprotease (MMP) cleavage site is a MMP 9 cleavage site, a MMP 13 cleavage site or a MMP 2 cleavage site. In embodiments, the disintegrin and metalloprotease domaincontaining (ADAM) metalloprotease cleavage site is a ADAM 9 metalloprotease cleavage site, a ADAM 10 metalloprotease cleavage site or a ADAM 17 metalloprotease cleavage site.

[0404] Further exemplary cleavage sites include the cleavage site of ABHD12, ADAM 12, ABHD12B, ABHD13, ABHD17A, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33, ADAM8, ABHD17A, ADAMDEC1, ADAMTS1, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS2, ADAMTS20, ADAMTS3, ADAMTS4, ABHD17B, ADAMTS5, Attorney Docket No.: 60846-0002W01

[0405] ADAMTS6, ADAMTS7, ADAMTS8, ADAMTS9, ADAMTSL I , ADAMTSL2, ADAMTSL3, ABHD17C, ADAMTSL5, ASTL, BMP1, CELA1, CELA2A, CELA2B, CELA3A, CELA3B, ADAMI 0, ADAMI 5, ADAMI 7, ADAM9, ADAMTS4, CTSE, CTSF, ADAMTSL4, CMA1, CTRB1, CTRC, CTSO, CTR1, CTSA, CTSW, CTSB, CTSC, CTSD, ESP1, CTSG, CTSH, GZMA, GZMB, GZMH, CTSK, GZMM, CTSL, CTSS, CTSV, CTSZ, HTRA4, KLK10, KLK11, KLK13, KLK14, KLK2, KLK4, DPP4, KLK6, KLK7, KLKB1, ECE1, ECE2, ECEL1, MASP2, MEP1A, MEP1B, ELANE, FAP, GZMA, MMP11, GZMK, HGFAC, HPN, HTRA1, MMP11, MMP16, MMP17, MMP19, HTRA2, MMP20, MMP21, HTRA3, HTRA4, KEL, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, KLK5, MMP3, MMP7, MMP8, MMP9, LGMN, LNPEP, MASP1, PAPP A, PAPPA2, PCSK1, NAPSA, PCSK5, PCSK6, MME, MMP1, MMP10, PLAT, PLAU, PLG, PRSS1, PRSS12, PRSS2, PRSS21, PRSS3, PRSS33, PRSS4, PRSS55, PRSS57, MMP12, PRSS8, PRSS9, PRTN3, MMP13, MMP14, STM, TMPRSS10, TMPRSS11A, TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS12, TMPRSS13, MMP15, TMPRSS15, MMP2, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS9, NRDC, OVCH1, PAMR1, PCSK3, PHEX, TINAG, TPSAB1, TPSD1, or TPSGL

[0406] In embodiments, the protease cleavable linker is a serum protease cleavable linker. In embodiments, the protease cleavable linker is a tumor microenvironment protease cleavable linker. In embodiments, the tumor microenvironment protease cleavable linker is a matrix metalloprotease (MMP) cleavable linker.

[0407] In embodiments, the protease cleavable linker includes the sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In embodiments, the protease cleavable linker includes the sequence of SEQ ID NO: 5. In embodiments, the protease cleavable linker includes the sequence of SEQ ID NO: 6. In embodiments, the protease cleavable linker includes the sequence of SEQ ID NO: 7. In embodiments, the protease cleavable linker includes the sequence of SEQ ID NO: 8. In embodiments, the protease cleavable linker includes the sequence of SEQ ID NO: 9. In embodiments, the protease cleavable linker includes the sequence of SEQ ID NO: 10. In embodiments, the protease cleavable linker includes the sequence of SEQ ID NO: 11. In embodiments, the protease cleavable linker includes the sequence of SEQ ID NO: 12. In some embodiments, the protease cleavage linker includes the sequence of SEQ ID NO: 557. In some embodiments, the protease cleavage linker includes the sequence of any one of SEQ ID NOs: 558-570. Attorney Docket No.: 60846-0002W01

[0408] In embodiments, the protease cleavable linker has the sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In embodiments, the protease cleavable linker has the sequence of SEQ ID NO: 5. In embodiments, the protease cleavable linker has the sequence of SEQ ID NO: 6. In embodiments, the protease cleavable linker has the sequence of SEQ ID NO: 7. In embodiments, the protease cleavable linker has the sequence of SEQ ID NO: 8. In embodiments, the protease cleavable linker has the sequence of SEQ ID NO: 9. In embodiments, the protease cleavable linker has the sequence of SEQ ID NO: 10. In embodiments, the protease cleavable linker has the sequence of SEQ ID NO: 11. In embodiments, the protease cleavable linker has the sequence of SEQ ID NO: 12. In some embodiments, the protease cleavage linker has the sequence of SEQ ID NO: 557. In some embodiments, the protease cleavage linker has the sequence of any one of SEQ ID NOs: 558- 570.

[0409] In some embodiments, the protease cleavable linker includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) GGGGS linkers (SEQ ID NO: 556). In some embodiments, the protease cleavable linker includes any one of the substrate sequences described herein. In some embodiments, the protease cleavable linker includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) GGGGS linkers flanking the substrate sequence. In some embodiments, the protease cleavable linker includes 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GGGGS linkers at the N- terminus of the substrate sequence. In some embodiments, the protease cleavable linker includes 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GGGGS linkers at the C-terminus of the substrate sequence.

[0410] Any of the protease cleavable linkers described in Zheng Su et al., Acta Pharm Sin B. 2021 Dec; 11(12): 3889-3907, which is hereby incorporated by reference in its entirety and for all purposes, may be used for the compositions and methods provided herein including embodiments thereof.

[0411] Any of the protease cleavable linkers described in Chen, E. I., Kridel, S. J., Howard, E. W., Li, W., Godzik, A., & Smith, J. W. (2002). A unique substrate recognition profile for matrix metalloproteinase-2. Journal of Biological Chemistry, 277(6), 4485-4491, which is hereby incorporated by reference in its entirety and for all purposes, may be used for the compositions and methods provided herein including embodiments thereof.

[0412] Any of the protease cleavable linkers described in Kridel, S. J., Chen, E., Kotra, L. P., Howard, E. W., Mobashery, S., & Smith, J. W. (2001). Substrate hydrolysis by matrix Attorney Docket No.: 60846-0002W01 metalloproteinase-9. Journal of Biological Chemistry, 276(23), 20572-20578, which is hereby incorporated by reference in its entirety and for all purposes, may be used for the compositions and methods provided herein including embodiments thereof.

[0413] Any of the protease cleavable linkers described in Lin, WW., Lu, YC., Chuang, CH. et al. Ab locks for improving the selectivity and safety of antibody drugs. J Biomed Sci 27, 76 (2020), which is hereby incorporated by reference in its entirety and for all purposes, may be used for the compositions and methods provided herein including embodiments thereof.

[0414] In embodiments, the protease cleavable linker has a length of about 2 to about 500 amino acids. In embodiments, the protease cleavable linker has a length of about 2 to about 450 amino acids. In embodiments, the protease cleavable linker has a length of about 2 to about 400 amino acids. In embodiments, the protease cleavable linker has a length of about 2 to about 350 amino acids. In embodiments, the protease cleavable linker has a length of about 2 to about 300 amino acids. In embodiments, the protease cleavable linker has a length of about 2 to about 250 amino acids. In embodiments, the protease cleavable linker has a length of about 2 to about 200 amino acids. In embodiments, the protease cleavable linker has a length of about 2 to about 150 amino acids. In embodiments, the protease cleavable linker has a length of about 2 to about 100 amino acids. In embodiments, the protease cleavable linker has a length of about 2 to about 50 amino acids.

[0415] In embodiments, the protease cleavable linker has a length of about 10 to about 500 amino acids. In embodiments, the protease cleavable linker has a length of about 10 to about 450 amino acids. In embodiments, the protease cleavable linker has a length of about 10 to about 400 amino acids. In embodiments, the protease cleavable linker has a length of about 10 to about 350 amino acids. In embodiments, the protease cleavable linker has a length of about 10 to about 300 amino acids. In embodiments, the protease cleavable linker has a length of about 10 to about 250 amino acids. In embodiments, the protease cleavable linker has a length of about 10 to about 200 amino acids. In embodiments, the protease cleavable linker has a length of about 10 to about 150 amino acids. In embodiments, the protease cleavable linker has a length of about 10 to about 100 amino acids. In embodiments, the protease cleavable linker has a length of about 10 to about 50 amino acids.

[0416] In embodiments, the protease cleavable linker has a length of about 15 to about 500 amino acids. In embodiments, the protease cleavable linker has a length of about 15 to about 450 amino acids. In embodiments, the protease cleavable linker has a length of about 15 to Attorney Docket No.: 60846-0002W01 about 400 amino acids. In embodiments, the protease cleavable linker has a length of about 15 to about 350 amino acids. In embodiments, the protease cleavable linker has a length of about 15 to about 300 amino acids. In embodiments, the protease cleavable linker has a length of about 15 to about 250 amino acids. In embodiments, the protease cleavable linker has a length of about 15 to about 200 amino acids. In embodiments, the protease cleavable linker has a length of about 15 to about 150 amino acids. In embodiments, the protease cleavable linker has a length of about 15 to about 100 amino acids. In embodiments, the protease cleavable linker has a length of about 15 to about 50 amino acids.

[0417] In embodiments, the protease cleavable linker has a length of about 20 to about 500 amino acids. In embodiments, the protease cleavable linker has a length of about 20 to about 450 amino acids. In embodiments, the protease cleavable linker has a length of about 20 to about 400 amino acids. In embodiments, the protease cleavable linker has a length of about 20 to about 350 amino acids. In embodiments, the protease cleavable linker has a length of about 20 to about 300 amino acids. In embodiments, the protease cleavable linker has a length of about 20 to about 250 amino acids. In embodiments, the protease cleavable linker has a length of about 20 to about 200 amino acids. In embodiments, the protease cleavable linker has a length of about 20 to about 150 amino acids. In embodiments, the protease cleavable linker has a length of about 20 to about 100 amino acids. In embodiments, the protease cleavable linker has a length of about 20 to about 50 amino acids.

[0418] In embodiments, the protease cleavable linker has a length of about 25 to about 500 amino acids. In embodiments, the protease cleavable linker has a length of about 25 to about 450 amino acids. In embodiments, the protease cleavable linker has a length of about 25 to about 400 amino acids. In embodiments, the protease cleavable linker has a length of about 25 to about 350 amino acids. In embodiments, the protease cleavable linker has a length of about 25 to about 300 amino acids. In embodiments, the protease cleavable linker has a length of about 25 to about 250 amino acids. In embodiments, the protease cleavable linker has a length of about 25 to about 200 amino acids. In embodiments, the protease cleavable linker has a length of about 25 to about 150 amino acids. In embodiments, the protease cleavable linker has a length of about 25 to about 100 amino acids. In embodiments, the protease cleavable linker has a length of about 25 to about 50 amino acids.

[0419] In embodiments, the protease cleavable linker has a length of about 30 to about 500 amino acids. In embodiments, the protease cleavable linker has a length of about 30 to about Attorney Docket No.: 60846-0002W01

[0420] 450 amino acids. In embodiments, the protease cleavable linker has a length of about 30 to about 400 amino acids. In embodiments, the protease cleavable linker has a length of about 30 to about 350 amino acids. In embodiments, the protease cleavable linker has a length of about 30 to about 300 amino acids. In embodiments, the protease cleavable linker has a length of about 30 to about 250 amino acids. In embodiments, the protease cleavable linker has a length of about 30 to about 200 amino acids. In embodiments, the protease cleavable linker has a length of about 30 to about 150 amino acids. In embodiments, the protease cleavable linker has a length of about 30 to about 100 amino acids. In embodiments, the protease cleavable linker has a length of about 30 to about 50 amino acids.

[0421] In embodiments, the protease cleavable linker has a length of about 35 to about 500 amino acids. In embodiments, the protease cleavable linker has a length of about 35 to about 450 amino acids. In embodiments, the protease cleavable linker has a length of about 35 to about 400 amino acids. In embodiments, the protease cleavable linker has a length of about 35 to about 350 amino acids. In embodiments, the protease cleavable linker has a length of about 35 to about 300 amino acids. In embodiments, the protease cleavable linker has a length of about 35 to about 250 amino acids. In embodiments, the protease cleavable linker has a length of about 35 to about 200 amino acids. In embodiments, the protease cleavable linker has a length of about 35 to about 150 amino acids. In embodiments, the protease cleavable linker has a length of about 35 to about 100 amino acids. In embodiments, the protease cleavable linker has a length of about 35 to about 50 amino acids.

[0422] In embodiments, the protease cleavable linker has a length of about 40 to about 500 amino acids. In embodiments, the protease cleavable linker has a length of about 40 to about 450 amino acids. In embodiments, the protease cleavable linker has a length of about 40 to about 400 amino acids. In embodiments, the protease cleavable linker has a length of about 40 to about 350 amino acids. In embodiments, the protease cleavable linker has a length of about 40 to about 300 amino acids. In embodiments, the protease cleavable linker has a length of about 40 to about 250 amino acids. In embodiments, the protease cleavable linker has a length of about 40 to about 200 amino acids. In embodiments, the protease cleavable linker has a length of about 40 to about 150 amino acids. In embodiments, the protease cleavable linker has a length of about 40 to about 100 amino acids. In embodiments, the protease cleavable linker has a length of about 40 to about 50 amino acids. Attorney Docket No.: 60846-0002W01

[0423] In embodiments, the protease cleavable linker has a length of about 45 to about 500 amino acids. In embodiments, the protease cleavable linker has a length of about 45 to about 450 amino acids. In embodiments, the protease cleavable linker has a length of about 45 to about 400 amino acids. In embodiments, the protease cleavable linker has a length of about 45 to about 350 amino acids. In embodiments, the protease cleavable linker has a length of about 45 to about 300 amino acids. In embodiments, the protease cleavable linker has a length of about 45 to about 250 amino acids. In embodiments, the protease cleavable linker has a length of about 45 to about 200 amino acids. In embodiments, the protease cleavable linker has a length of about 45 to about 150 amino acids. In embodiments, the protease cleavable linker has a length of about 45 to about 100 amino acids. In embodiments, the protease cleavable linker has a length of about 45 to about 50 amino acids.

[0424] In embodiments, the protease cleavable linker has a length of about 2, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids.

[0425] In embodiments, the protease cleavable linker has a length of 2 to 500 amino acids. In embodiments, the protease cleavable linker has a length of 2 to 450 amino acids. In embodiments, the protease cleavable linker has a length of 2 to 400 amino acids. In embodiments, the protease cleavable linker has a length of 2 to 350 amino acids. In embodiments, the protease cleavable linker has a length of 2 to 300 amino acids. In embodiments, the protease cleavable linker has a length of 2 to 250 amino acids. In embodiments, the protease cleavable linker has a length of 2 to 200 amino acids. In embodiments, the protease cleavable linker has a length of 2 to 150 amino acids. In embodiments, the protease cleavable linker has a length of 2 to 100 amino acids. In embodiments, the protease cleavable linker has a length of 2 to 50 amino acids.

[0426] In embodiments, the protease cleavable linker has a length of 10 to 500 amino acids. In embodiments, the protease cleavable linker has a length of 10 to 450 amino acids. In embodiments, the protease cleavable linker has a length of 10 to 400 amino acids. In embodiments, the protease cleavable linker has a length of 10 to 350 amino acids. In embodiments, the protease cleavable linker has a length of 10 to 300 amino acids. In embodiments, the protease cleavable linker has a length of 10 to 250 amino acids. In embodiments, the protease cleavable linker has a length of 10 to 200 amino acids. In embodiments, the protease cleavable linker has a length of 10 to 150 amino acids. In Attorney Docket No.: 60846-0002W01 embodiments, the protease cleavable linker has a length of 10 to 100 amino acids. In embodiments, the protease cleavable linker has a length of 10 to 50 amino acids.

[0427] In embodiments, the protease cleavable linker has a length of 15 to 500 amino acids. In embodiments, the protease cleavable linker has a length of 15 to 450 amino acids. In embodiments, the protease cleavable linker has a length of 15 to 400 amino acids. In embodiments, the protease cleavable linker has a length of 15 to 350 amino acids. In embodiments, the protease cleavable linker has a length of 15 to 300 amino acids. In embodiments, the protease cleavable linker has a length of 15 to 250 amino acids. In embodiments, the protease cleavable linker has a length of 15 to 200 amino acids. In embodiments, the protease cleavable linker has a length of 15 to 150 amino acids. In embodiments, the protease cleavable linker has a length of 15 to 100 amino acids. In embodiments, the protease cleavable linker has a length of 15 to 50 amino acids.

[0428] In embodiments, the protease cleavable linker has a length of 20 to 500 amino acids. In embodiments, the protease cleavable linker has a length of 20 to 450 amino acids. In embodiments, the protease cleavable linker has a length of 20 to 400 amino acids. In embodiments, the protease cleavable linker has a length of 20 to 350 amino acids. In embodiments, the protease cleavable linker has a length of 20 to 300 amino acids. In embodiments, the protease cleavable linker has a length of 20 to 250 amino acids. In embodiments, the protease cleavable linker has a length of 20 to 200 amino acids. In embodiments, the protease cleavable linker has a length of 20 to 150 amino acids. In embodiments, the protease cleavable linker has a length of 20 to 100 amino acids. In embodiments, the protease cleavable linker has a length of 20 to 50 amino acids.

[0429] In embodiments, the protease cleavable linker has a length of 25 to 500 amino acids. In embodiments, the protease cleavable linker has a length of 25 to 450 amino acids. In embodiments, the protease cleavable linker has a length of 25 to 400 amino acids. In embodiments, the protease cleavable linker has a length of 25 to 350 amino acids. In embodiments, the protease cleavable linker has a length of 25 to 300 amino acids. In embodiments, the protease cleavable linker has a length of 25 to 250 amino acids. In embodiments, the protease cleavable linker has a length of 25 to 200 amino acids. In embodiments, the protease cleavable linker has a length of 25 to 150 amino acids. In embodiments, the protease cleavable linker has a length of 25 to 100 amino acids. In embodiments, the protease cleavable linker has a length of 25 to 50 amino acids. Attorney Docket No.: 60846-0002W01

[0430] In embodiments, the protease cleavable linker has a length of 30 to 500 amino acids. In embodiments, the protease cleavable linker has a length of 30 to 450 amino acids. In embodiments, the protease cleavable linker has a length of 30 to 400 amino acids. In embodiments, the protease cleavable linker has a length of 30 to 350 amino acids. In embodiments, the protease cleavable linker has a length of 30 to 300 amino acids. In embodiments, the protease cleavable linker has a length of 30 to 250 amino acids. In embodiments, the protease cleavable linker has a length of 30 to 200 amino acids. In embodiments, the protease cleavable linker has a length of 30 to 150 amino acids. In embodiments, the protease cleavable linker has a length of 30 to 100 amino acids. In embodiments, the protease cleavable linker has a length of 30 to 50 amino acids.

[0431] In embodiments, the protease cleavable linker has a length of 35 to 500 amino acids. In embodiments, the protease cleavable linker has a length of 35 to 450 amino acids. In embodiments, the protease cleavable linker has a length of 35 to 400 amino acids. In embodiments, the protease cleavable linker has a length of 35 to 350 amino acids. In embodiments, the protease cleavable linker has a length of 35 to 300 amino acids. In embodiments, the protease cleavable linker has a length of 35 to 250 amino acids. In embodiments, the protease cleavable linker has a length of 35 to 200 amino acids. In embodiments, the protease cleavable linker has a length of 35 to 150 amino acids. In embodiments, the protease cleavable linker has a length of 35 to 100 amino acids. In embodiments, the protease cleavable linker has a length of 35 to 50 amino acids.

[0432] In embodiments, the protease cleavable linker has a length of 40 to 500 amino acids. In embodiments, the protease cleavable linker has a length of 40 to 450 amino acids. In embodiments, the protease cleavable linker has a length of 40 to 400 amino acids. In embodiments, the protease cleavable linker has a length of 40 to 350 amino acids. In embodiments, the protease cleavable linker has a length of 40 to 300 amino acids. In embodiments, the protease cleavable linker has a length of 40 to 250 amino acids. In embodiments, the protease cleavable linker has a length of 40 to 200 amino acids. In embodiments, the protease cleavable linker has a length of 40 to 150 amino acids. In embodiments, the protease cleavable linker has a length of 40 to 100 amino acids. In embodiments, the protease cleavable linker has a length of 40 to 50 amino acids.

[0433] In embodiments, the protease cleavable linker has a length of 45 to 500 amino acids. In embodiments, the protease cleavable linker has a length of 45 to 450 amino acids. In Attorney Docket No.: 60846-0002W01 embodiments, the protease cleavable linker has a length of 45 to 400 amino acids. In embodiments, the protease cleavable linker has a length of 45 to 350 amino acids. In embodiments, the protease cleavable linker has a length of 45 to 300 amino acids. In embodiments, the protease cleavable linker has a length of 45 to 250 amino acids. In embodiments, the protease cleavable linker has a length of 45 to 200 amino acids. In embodiments, the protease cleavable linker has a length of 45 to 150 amino acids. In embodiments, the protease cleavable linker has a length of 45 to 100 amino acids. In embodiments, the protease cleavable linker has a length of 45 to 50 amino acids. In embodiments, the protease cleavable linker has a length of 45 to 50 amino acids.

[0434] In embodiments, the protease cleavable linker has a length of 2, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids.

[0435] In embodiments, the protease cleavable linker has a length of about 5 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 6 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 7 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 8 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 9 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 10 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 11 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 12 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 13 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 14 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 15 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 16 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 17 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 18 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 19 to about 20 amino acids. In embodiments, the protease cleavable linker has a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0436] In embodiments, the protease cleavable linker has a length of 5 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 6 to 20 amino acids. In Attorney Docket No.: 60846-0002W01 embodiments, the protease cleavable linker has a length of 7 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 8 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 9 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 10 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 11 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 12 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 13 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 14 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 15 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 16 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 17 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 18 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 19 to 20 amino acids. In embodiments, the protease cleavable linker has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0437] In embodiments, the protease cleavable linker has a length of about 5 to about 19 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 18 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 17 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 16 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 15 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 14 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 13 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 12 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 11 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 10 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 9 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 8 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 7 amino acids. In embodiments, the protease cleavable linker has a length of about 5 to about 6 amino acids.

[0438] In embodiments, the protease cleavable linker has a length of 5 to 19 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 18 amino acids. In Attorney Docket No.: 60846-0002W01 embodiments, the protease cleavable linker has a length of 5 to 17 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 16 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 15 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 14 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 13 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 12 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 11 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 10 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 9 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 8 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 7 amino acids. In embodiments, the protease cleavable linker has a length of 5 to 6 amino acids.

[0439] In embodiments, the protease is a serum protease or a tumor microenvironment protease. In embodiments, the protease is a serum protease. In embodiments, the protease is a tumor microenvironment protease. In embodiments, the protease is a matrix metalloprotease, an ADAM protease, a caspase protease, a PSA protease, a calpain protease, a legumain protease, a Fap protease, a DPPIV protease, or a PEP protease. In embodiments, the protease is a matrix metalloprotease. In embodiments, the protease is an ADAM protease. In embodiments, the protease is a caspase protease. In embodiments, the protease is a PSA protease. In embodiments, the protease is a calpain protease. In embodiments, the protease is a legumain protease. In embodiments, the protease is a Fap protease. In embodiments, the protease is a DPPIV protease. In embodiments, the protease is a PEP protease.

[0440] In embodiments, the tumor microenvironment protease is MMP-2, MMP-9 or MMP- 14. In embodiments, the tumor microenvironment protease is MMP-2. In embodiments, the tumor microenvironment protease is MMP-9. In embodiments, the tumor microenvironment protease is MMP-14. In embodiments, the tumor microenvironment protease is MMP-2 / 9.

[0441] In embodiments, the serum protease is a thrombin protease, a plasmin protease, a trypsin protease, a chymotrypsin protease, a factor Xa protease, a factor Xlla protease, a factor Xia protease, a factor IXa protease, a kallikrein protease, a complement Cis protease, a complement Clr protease, a cathepsin protease, an elastase, a carboxypeptidase, an angiotensin-converting enzyme (ACE), a granzyme protease, or a MASP1 protease. In embodiments, the serum protease is a thrombin protease. In embodiments, the serum protease Attorney Docket No.: 60846-0002W01 is a plasmin protease. In embodiments, the serum protease is a trypsin protease. In embodiments, the serum protease is a chymotrypsin protease. In embodiments, the serum protease is a factor Xa protease. In embodiments, the serum protease is a factor Xlla protease. In embodiments, the serum protease is a factor Xia protease. In embodiments, the serum protease is a factor IXa protease. In embodiments, the serum protease is a kallikrein protease. In embodiments, the serum protease is a complement Cis protease. In embodiments, the serum protease is a complement Clr protease. In embodiments, the serum protease is a cathepsin protease. In embodiments, the serum protease is an elastase. In embodiments, the serum protease is a carboxypeptidase. In embodiments, the serum protease is an angiotensinconverting enzyme (ACE). In embodiments, the serum protease is a granzyme protease. In embodiments, the serum protease is a MASP1 protease.

[0442] The Fc dimerizing domain provided herein including embodiments thereof is covalently bound to at least one therapeutic moiety. In embodiments, a chemical linker binds the at least one therapeutic moiety to the Fc dimerizing domain. Any of the linkers and therapeutic moieties described in Pharmaceutics. 2022 Feb; 14(2): 396, which is hereby incorporated by reference in its entirety and for all purposes, may be used for the compositions and methods provided herein including embodiments thereof. Further, any of the antibody domains, linkers and therapeutic moieties described in Nature Reviews Clinical Oncology volume 21, pages 203-223 (2024), which is hereby incorporated by reference in its entirety and for all purposes, may be used for the compositions and methods provided herein including embodiments thereof.

[0443] In embodiments, the at least one therapeutic moiety is a small molecule, a peptide, or a nucleic acid. In embodiments, the at least one therapeutic moiety is a small molecule. In embodiments, the at least one therapeutic moiety is a peptide. In embodiments, the at least one therapeutic moiety is a nucleic acid. In embodiments, the at least one therapeutic moiety is a microtubule targeting therapeutic moiety, a DNA damaging therapeutic moiety, an RNA polymerase inhibiting therapeutic moiety, or a topoisomerase inhibiting therapeutic moiety. In embodiments, the at least one therapeutic moiety is a microtubule targeting therapeutic moiety. In embodiments, the at least one therapeutic moiety is a DNA damaging therapeutic moiety. In embodiments, the at least one therapeutic moiety is an RNA polymerase inhibiting therapeutic moiety. In embodiments, the at least one therapeutic moiety is a topoisomerase inhibiting therapeutic moiety. Attorney Docket No.: 60846-0002W01

[0444] In embodiments, the at least one therapeutic moiety is a plurality of therapeutic moieties. A plurality of therapeutic moieties as provided herein refers to more than one therapeutic moiety (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 200, 500, 1000, 2000, 5000 etc.).

[0445] The recombinant polypeptides provided herein may include multiple domains (e.g., a cancer antigen binding domain, an effector cell binding domain, an Fc dimerizing domain and / or a protease cleavable linker) all of which form part of one continuous single chain polypeptide. The continuous single chain polypeptide as provided herein may be a polypeptide chain including multiple domains (e.g., a cancer antigen binding domain, an effector cell binding domain, an Fc dimerizing domain and / or a protease cleavable linker) covalently attached to each other thereby forming said continuous polypeptide chain. In embodiments, the recombinant polypeptide includes a cancer antigen binding domain covalently bound to an effector cell binding domain; an Fc dimerizing domain covalently bound to at least one therapeutic moiety; and a protease cleavable linker covalently binding the cancer antigen binding domain to the Fc dimerizing domain through covalently binding the effector cell binding domain to the Fc dimerizing domain. Thus, in embodiments, the polypeptide is a single chain polypeptide.

[0446] In embodiments, the recombinant polypeptide includes from the N-terminus to the C- terminus cancer antigen binding domain, a chemical linker, an effector cell binding domain, a protease cleavable linker and an Fc dimerizing domain covalently bound to at least one therapeutic moiety. In embodiments, the cancer antigen binding domain is a DLL3 binding domain and the effector cell binding domain is a CD3 binding domain.

[0447] In embodiments, the cancer antigen binding domain is a NECTIN-4 binding domain and said effector cell binding domain is a CD3 binding domain.

[0448] In embodiments, the at least one therapeutic moiety is Dxd, Exatecan, TM (trastuzumab maytansinoid), or MMAE. In embodiments, the at least one therapeutic moiety is Dxd. In embodiments, the at least one therapeutic moiety is TM. In embodiments, the at least one therapeutic moiety is MMAE. In embodiments, the at least one therapeutic moiety is Exatecan.

[0449] Any of the therapeutic moieties described in Acta Pharmaceutica Sinica B Volume 13, Issue 10 , October 2023, Pages 4025-4059; J. Med. Chem. 2023, 66, 1, 140-148; Medicine in Drug Discovery Volume 15, September 2022, 100128; and Signal Transduction and Targeted Therapy volume 7, Article number: 20 (2022); which are incorporated by Attorney Docket No.: 60846-0002W01 reference in their entirety and for all purposes, may be used for the compositions and methods provided herein including embodiments thereof.

[0450] In one embodiment, the recombinant polypeptide includes from the N-terminus to the C -terminus a DLL3 binding domain, a CD3 binding domain, a protease cleavable link...

Claims

Attorney Docket No.: 60846-0002W01WHAT IS CLAIMED IS:

1. A protein complex comprising:(a) a fragment crystallizable (Fc) region;(b) a first antigen-binding moiety that specifically binds to a first cancer antigen; and(c) a second antigen-binding moiety that specifically binds to an effector cell antigen, wherein the second antigen-binding moiety is linked to the Fc region via a first linker peptide.

2. The protein complex of claim 1, wherein the first antigen-binding moiety is linked to the second antigen-binding moiety.

3. The protein complex of claim 1 or 2, wherein at least one therapeutic agent is conjugated to the Fc region, optionally the at least one therapeutic agent is a cytotoxic or cytostatic agent.

4. The protein complex of any one of claims 1-3, wherein the first linker peptide is cleavable by a protease within tumor microenvironment.

5. The protein complex of claim 4, wherein the first antigen-binding moiety and the second antigen-binding moiety form a T cell engager (TCE) when the first linker peptide is cleaved.

6. The protein complex of any one of claims 1-5, wherein the first antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or multi-specific antibody (e.g., a bispecific antibody).

7. The protein complex of any one of claims 1-6, wherein the first cancer antigen is CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD96, CD123, CDH17, CEA, CLDN6, CLDN18.2, cMET, CCR4, DLL3, DLL4, DKK, NECTIN4, Claudin, LIVE6, BCMA, HER2, HER3, HLA-G, KLK2, LGR5, MAGEA4 / 8, mesothelin, angiopoietin, FGF, FGFR, MUC16, PRAME, PSMA, STEAP1, TMEFF2, PD-L1, EGFR, EGFRvIII, ENPP3, EpCAM, gplOO, GPC3, GUCY2C, VEGF, VEGFR2, CTLA-4, PD-1, RANK-L, GD2, SLAMF7, PDGFRA, TROP-2, FR-alpha, FcRH5, FLT3, GPRC5D, R0R1, 5T4, B7-H3, or B7-H4.Attorney Docket No.: 60846-0002W018. The protein complex of any one of claims 1-7, wherein the second antigen -binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or a multi-specific antibody (e.g., a bispecific antibody).

9. The protein complex of any one of claims 1-8, wherein the effector cell antigen is CD3, CD28, PD-1, TIGIT, Lag3, CD16A, CD16B, CD32A, CD32B, CD32C, CD64, or SIRPa.

10. The protein complex of any one of claims 1-9, wherein the Fc region comprises:(a) a first polypeptide comprising an optional first hinge region, a first CH2 domain, and a first CH3 domain, and(b) a second polypeptide comprising an optional second hinge region, a second CH2 domain, and a second CH3 domain, wherein the first and second polypeptides associate with each other, forming the Fc region.

11. The protein complex of any one of claims 1-10, further comprising: a third antigenbinding moiety that specifically binds to a second cancer antigen, and a fourth antigen-binding moiety that specifically binds to the effector cell antigen, wherein the fourth antigen-binding moiety is linked to the Fc region via a second linker peptide.

12. The protein complex of claim 11, wherein the third antigen-binding moiety is linked to the fourth antigen-binding moiety.

13. The protein complex of claim 11 or 12, wherein the second linker peptide is cleavable by a protease within tumor microenvironment.

14. The protein complex of any one of claims 11-13, wherein the third antigen-binding moiety and the fourth antigen-binding moiety form a T cell engager (TCE) when the second linker peptide is cleaved.

15. The protein complex of any one of claims 11-14, wherein the third antigen -binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or multi-specific antibody (e.g., a bispecific antibody).Attorney Docket No.: 60846-0002W0116. The protein complex of any one of claims 11-15, wherein the second cancer antigen is CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD96, CD123, CDH17, CEA, CLDN6, CLDN18.2, cMET, CCR4, DLL3, DLL4, DKK, NECTIN4, Claudin, LIVE6, BCMA, HER2, HER3, HLA-G, KLK2, LGR5, MAGEA4 / 8, mesothelin, angiopoietin, FGF, FGFR, MUC16, PRAME, PSMA, STEAP1, TMEFF2, PD-L1, EGFR, EGFRvIII, ENPP3, EpCAM, gplOO, GPC3, GUCY2C, VEGF, VEGFR2, CTLA-4, PD-1, RANK-L, GD2, SLAMF7, PDGFRA, TROP-2, FR-alpha, FcRH5, FLT3, GPRC5D, R0R1, 5T4, B7-H3, or B7-H4.

17. The protein complex of any one of claims 11-16, wherein the fourth antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or a multi-specific antibody (e.g., a bispecific antibody).

18. The protein complex of any one of claims 11-17, wherein the first cancer antigen and the second cancer antigen are the same.

19. The protein complex of any one of claims 11-18, wherein the first and third antigenbinding moieties are a VHH, and / or wherein the second and fourth antigen-binding moieties are an scFv.

20. The protein complex of any one of claims 11-19, further comprising: a fifth antigenbinding moiety that specifically binds to a third cancer antigen, and a sixth antigenbinding moiety that specifically binds to a fourth cancer antigen; optionally the fifth antigen-binding moiety is linked to the first antigen-binding moiety, and the sixth antigen-binding moiety is linked to the third antigen-binding moiety.

21. The protein complex of claim 20, wherein the fifth and / or sixth antigen-binding moieties are an scFv, a Fab, a single domain antibody (e.g., a VHH), or multi-specific antibody (e.g., a bispecific antibody).

22. The protein complex of claim 20 or 21, wherein the first, third, fifth and sixth antigenbinding moieties are a VHH, and / or wherein the second and fourth antigen-binding moieties are an scFv.Attorney Docket No.: 60846-0002W0123. The protein complex of any one of claims 1-10, further comprising: a third antigenbinding moiety that specifically binds to a second cancer antigen; wherein the third antigen-binding moiety is linked to the Fc region.

24. The protein complex of claim 23, wherein the third antigen-binding moiety and the Fc region form an antibody-drug conjugate (ADC) when the first linker peptide is cleaved.

25. The protein complex of claim 23 or 24, wherein the third antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or multi-specific antibody (e.g., a bispecific antibody).

26. The protein complex of any one of claims 23-25, wherein the third cancer antigen is CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD96, CD123, CDH17, CEA, CLDN6, CLDN18.2, cMET, CCR4, DLL3, DLL4, DKK, NECTIN4, Claudin, LIVE6, BCMA, HER2, HER3, HLA-G, KLK2, LGR5, MAGEA4 / 8, mesothelin, angiopoietin, FGF, FGFR, MUC16, PRAME, PSMA, STEAP1, TMEFF2, PD-L1, EGFR, EGFRvIII, ENPP3, EpCAM, gplOO, GPC3, GUCY2C, VEGF, VEGFR2, CTLA-4, PD-1, RANK-L, GD2, SLAMF7, PDGFRA, TROP-2, FR-alpha , FcRH5, FLT3, GPRC5D, ROR1, 5T4, B7-H3, or B7-H4.

27. The protein complex of any one of claims 23-26, wherein the first cancer antigen and the second cancer antigen are the same.

28. The protein complex of any one of claims 23-27, wherein the first antigen -binding moiety is a VHH, and / or wherein the second and third antigen-binding moieties are an scFv.

29. The protein complex of any one of claims 23-28, further comprising: a fourth antigenbinding moiety that specifically binds to a third cancer antigen; wherein the fourth antigen-binding moiety is linked to the first antigen-binding moiety.

30. The protein complex of claim 29, wherein the fourth antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or multi-specific antibody (e.g., a bispecific antibody).Attomey Docket No.: 60846-0002W0131. The protein complex of claim 29 or 30, wherein the first and fourth antigen-binding moieties are a VHH, and / or wherein the second and third antigen-binding moieties are an scFv.

32. The protein complex of any one of claims 23-27, further comprising: a fourth antigenbinding moiety that specifically binds to a third cancer antigen; wherein the fourth antigen-binding moiety is linked to the third antigen-binding moiety.

33. The protein complex of claim 32, wherein the fourth antigen-binding moiety is an scFv, a Fab, a single domain antibody (e.g., a VHH), or multi-specific antibody (e.g., a bispecific antibody).

34. The protein complex of claim 32 or 33, wherein the first and second antigen-binding moieties are an scFv, and / or wherein the third and fourth antigen-binding moieties are a VHH.

35. The protein complex of any one of claims 1-34, wherein the effector cell antigen is CD3.

36. The protein complex of any one of claims 1-35, wherein the Fc region does not induce antibody effector functions, e.g., antibody-dependent cellular cytotoxicity (ADCC).

37. A protein complex comprising:(a) a fragment crystallizable (Fc) region, wherein at least one therapeutic agent is conjugated to the Fc region;(b) a first antigen-binding moiety that specifically binds to a first cancer antigen and a second antigen-binding moiety that specifically binds to an effector cell antigen, wherein the first antigen-binding moiety is linked to the second antigen-binding moiety, wherein the second antigen-binding moiety is linked to the Fc region via a first linker peptide; and(c) a third antigen-binding moiety that specifically binds to a second cancer antigen and a fourth antigen-binding moiety that specifically binds to the effector cell antigen, wherein the third antigen-binding moiety is linked to the fourth antigen-Attorney Docket No.: 60846-0002W01 binding moiety, wherein the fourth antigen-binding moiety is linked to the Fc region via a second linker peptide; wherein the first antigen-binding moiety and the second antigen-binding moiety form a first T cell engager (TCE) when the first linker peptide is cleaved, and wherein the third antigen-binding moiety and the fourth antigen-binding moiety form a second TCE when the second linker peptide is cleaved.

38. A protein complex comprising:(a) a fragment crystallizable (Fc) region, wherein at least one therapeutic agent is conjugated to the Fc region;(b) a first antigen-binding moiety that specifically binds to a first cancer antigen and a second antigen-binding moiety that specifically binds to an effector cell antigen, wherein the first antigen-binding moiety is linked to the second antigen-binding moiety, wherein the second antigen-binding moiety is linked to the Fc region via a first linker peptide; and(c) a third antigen-binding moiety that specifically binds to a second cancer antigen, wherein the third antigen-binding moiety is linked to the Fc; wherein when the first linker peptide is cleaved, the first antigen-binding moiety and the second antigen-binding moiety form a T cell engager (TCE), and the third antigen-binding and the Fc region form an antibody-drug conjugate (ADC).

39. The protein complex of claim 37 or 38, wherein the protein complex forms an ADC before the first and / or second linker peptides are cleaved.

40. A protein complex comprising:(a) a first polypeptide comprising, optionally form N-terminus to C-terminus:(1) a first antigen-binding moiety that specifically binds to a first cancer antigen,(2) a second antigen-binding moiety that specifically binds to a first effector cell antigen,(3) a first linker peptide,(4) an optional first hinge region,(5) a first CH2 domain, andAttorney Docket No.: 60846-0002W01(6) a first CH3 domain;(b) a second polypeptide comprising, optionally form N-terminus to C-terminus:(1) a third antigen-binding moiety that specifically binds to a second cancer antigen,(2) a fourth antigen-binding moiety that specifically binds to a second effector cell antigen,(3) a second linker peptide,(4) an optional second hinge region,(5) a second CH2 domain, and(6) a second CH3 domain; wherein the first and second linker peptides are cleavable by a protease within tumor microenvironment.

41. The protein complex of claim 40, further comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) therapeutic agent conjugated to the first and / or second polypeptides.

42. The protein complex of claim 40 or 41, wherein the first antigen-binding moiety and the second antigen-binding moiety form a first T cell engager (TCE) when the first linker peptide is cleaved, and wherein the third antigen-binding moiety and the fourth antigen-binding moiety form a second T cell engager (TCE) when the second linker peptide is cleaved.

43. A protein complex comprising:(a) a first polypeptide comprising, optionally form N-terminus to C-terminus:(1) a first antigen-binding moiety that specifically binds to a first cancer antigen,(2) a second antigen-binding moiety that specifically binds to a second cancer antigen,(3) a third antigen-binding moiety that specifically binds to a first effector cell antigen,(4) a first linker peptide,(5) an optional first hinge region,(6) a first CH2 domain, and(7) a first CH3 domain; andAttorney Docket No.: 60846-0002W01(b) a second polypeptide comprising, optionally form N-terminus to C-terminus:(1) a fourth antigen-binding moiety that specifically binds to a third cancer antigen,(2) a fifth antigen-binding moiety that specifically binds to a fourth cancer antigen,(3) a sixth antigen-binding moiety that specifically binds to a second effector cell antigen,(4) a second linker peptide,(5) an optional second hinge region,(6) a second CH2 domain, and(7) a second CH3 domain; wherein the first and second linker peptides are cleavable by a protease within tumor microenvironment.

44. The protein complex of claim 43, further comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) therapeutic agent conjugated to the first and / or second polypeptides.

45. The protein complex of claim 43 or 44, wherein the first, second, and third antigenbinding moieties form a first T cell engager (TCE) when the first linker peptide is cleaved, and wherein the fourth, fifth, and sixth antigen-binding moieties form a second T cell engager (TCE) when the second linker peptide is cleaved.

46. A protein complex comprising:(a) a first polypeptide comprising, optionally form N-terminus to C-terminus:(1) a first antigen-binding moiety that specifically binds to a first cancer antigen,(2) a second antigen-binding moiety that specifically binds to an effector cell antigen,(3) a first linker peptide,(4) an optional first hinge region,(5) a first CH2 domain, and(6) a first CH3 domain; and(b) a second polypeptide comprising, optionally form N-terminus to C-terminus:Attorney Docket No.: 60846-0002W01(1) a third antigen-binding moiety that specifically binds to a second cancer antigen,(2) an optional second hinge region,(3) a second CH2 domain, and(4) a second CH3 domain; wherein the first linker peptide is cleavable by a protease within tumor microenvironment.

47. The protein complex of claim 46, further comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) therapeutic agent conjugated to the first and / or second polypeptides.

48. The protein complex of claim 46 or 47, wherein when the first linker peptide is cleaved, the first antigen-binding moiety and the second antigen-binding moiety form a T cell engager (TCE) and the third antigen-binding moiety, the optional first hinge region, the optional second hinge region, the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain together form an antibody-drug conjugate (ADC).

49. A protein complex comprising:(a) a first polypeptide comprising, optionally form N-terminus to C-terminus:(1) a first antigen-binding moiety that specifically binds to a first cancer antigen,(2) a second antigen-binding moiety that specifically binds to a second cancer antigen,(3) a third antigen-binding moiety that specifically binds to an effector cell antigen,(4) a first linker peptide,(5) an optional first hinge region,(6) a first CH2 domain, and(7) a first CH3 domain; and(b) a second polypeptide comprising, optionally form N-terminus to C-terminus:(1) a fourth antigen-binding moiety that specifically binds to a third cancer antigen,(2) an optional second hinge region,Attorney Docket No.: 60846-0002W01(3) a second CH2 domain, and(4) a second CH3 domain; wherein the first linker peptide is cleavable by a protease within tumor microenvironment.

50. The protein complex of claim 49, further comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) therapeutic agent conjugated to the first and / or second polypeptides.

51. The protein complex of claim 49 or 50, wherein when the first linker peptide is cleaved, the first, second, and third antigen-binding moieties form a T cell engager (TCE) and the fourth antigen-binding moiety, the optional first hinge region, the optional second hinge region, the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain together form an antibody-drug conjugate (ADC).

52. A protein complex comprising:(a) a first polypeptide comprising, optionally form N-terminus to C-terminus:(1) a first antigen-binding moiety that specifically binds to a first cancer antigen,(2) a second antigen-binding moiety that specifically binds to an effector cell antigen,(3) a first linker peptide,(4) an optional first hinge region,(5) a first CH2 domain, and(6) a first CH3 domain; and(b) a second polypeptide comprising, optionally form N-terminus to C-terminus:(1) a third antigen-binding moiety that specifically binds to a second cancer antigen,(2) a fourth antigen-binding moiety that specifically binds to a third cancer antigen,(3) an optional second hinge region,(4) a second CH2 domain, and(5) a second CH3 domain;Attorney Docket No.: 60846-0002W01 wherein the first linker peptide is cleavable by a protease within tumor microenvironment.

53. The protein complex of claim 52, further comprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) therapeutic agent conjugated to the first and / or second polypeptides.

54. The protein complex of claim 52 or 53, wherein when the first linker peptide is cleaved, the first and second antigen-binding moieties form a T cell engager (TCE), and the third antigen-binding moiety, the fourth antigen-binding moiety, the optional first hinge region, the optional second hinge region, the first CH2 domain, the second CH2 domain, the first CH3 domain, and the second CH3 domain together form an antibody-drug conjugate (ADC).

55. A nucleic acid comprising a polynucleotide encoding the protein complex of any one of claims 1-54.

56. The nucleic acid of claim 55, wherein the nucleic acid is a DNA (e.g., cDNA) or RNA (e.g., mRNA).

57. A vector comprising one or more of the nucleic acids of claim 55 or 56.

58. A cell comprising the vector of claim 57.

59. The cell of claim 58, wherein the cell is a CHO cell.

60. A cell comprising the nucleic acid of claim 55 or 56.

61. A method of producing a protein complex, the method comprising(1) culturing the cell of any one of claims 58-60 under conditions sufficient for the cell to produce the protein complex; and(2) collecting the protein complex produced by the cell.Attorney Docket No.: 60846-0002W0162. A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the protein complex of any one of claims 1-54, to the subject.

63. The method of claim 62, wherein the subject has a cancer cell expressing a cancer antigen (e.g., DLL3).

64. The method of claim 62 or 63, wherein the cancer is breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, diffuse large B-cell lymphoma, mesothelioma, lung cancer, ovarian cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, cholangiocarcinoma, head and neck cancer, blood cancer, or a combination thereof.

65. A method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the protein complex of any one of claims 1-54.

66. A method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the protein complex of any one of claims 1-54.

67. A pharmaceutical composition comprising the protein complex of any one of claims 1-54, and a pharmaceutically acceptable carrier.

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