DLL3-Targeted Trispecific Proteins and Methods of Use Thereof

Abstract

Provided herein are DLL3 binding proteins and DLL3-targeting multispecific proteins (e.g., DLL3-targeting trispecific proteins) that include a domain that binds to CD3, a half-life extending domain, and a domain that binds to DLL3 (such as the DLL3-binding proteins provided herein). Also provided are pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making the DLL3 binding proteins, DLL3-targeting trispecific proteins. Also disclosed are methods of using the disclosed DLL3 binding proteins, DLL3-targeting trispecific proteins in the prevention and / or treatment of diseases, conditions, and disorders.

Description

[Technical field]

[0001] cross reference This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 196,619, filed June 3, 2021, U.S. Provisional Patent Application No. 63 / 288,939, filed December 13, 2021, and U.S. Provisional Patent Application No. 63 / 345,150, filed May 24, 2022, each of which is incorporated by reference in its entirety. [Background technology]

[0002] The selective destruction of individual cells or specific cell types is often desired in various clinical settings. For example, the specific destruction of tumor cells while leaving healthy cells and tissues intact and undamaged is a major goal of cancer therapy. One such method is by eliciting an immune response against the tumor to induce immune effector cells, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs), to attack and destroy tumor cells. Summary of the Invention

[0003] Described herein are methods of treating cancer, the methods comprising administering to a subject an effective amount of a delta-like ligand 3 (DLL3) targeting trispecific protein, wherein the protein comprises (a) a first domain (A) that specifically binds to human CD3, (b) a second domain (B) that is a half-life extending domain, and (c) a third domain (C) that specifically binds to DLL3, wherein the DLL3 targeting trispecific protein is administered at a dosage of about 1 μg to about 100 mg. In some embodiments, the DLL3 targeting trispecific protein is administered at a dosage of about 1 μg to about 14 mg. In some embodiments, the DLL3 targeting trispecific protein is administered at a dosage of about 1 μg to about 5 mg. In some embodiments, the DLL3 targeting trispecific protein is administered at a dosage of about 1 μg to about 2 mg. In some embodiments, the DLL3 targeting trispecific protein is administered at a dosage of about 1 μg to about 1 mg. In some embodiments, the DLL3 targeting trispecific protein is administered in a dosage of about 15 μg to about 3600 mg. In some embodiments, the DLL3 targeting trispecific protein is administered in a dosage of about 15 μg. In some embodiments, the DLL3 targeting trispecific protein is administered in a dosage of about 45 μg. In some embodiments, the DLL3 targeting trispecific protein is administered in a dosage of about 135 μg. In some embodiments, the DLL3 targeting trispecific protein is administered in a dosage of about 405 μg. In some embodiments, the DLL3 targeting trispecific protein is administered in a dosage of about 1215 μg. In some embodiments, the DLL3 targeting trispecific protein is administered in a dosage of about 3600 μg. In some embodiments, the DLL3 targeting trispecific protein is administered in a dosage of about 5 mg. In some embodiments, the DLL3 targeting trispecific protein is administered in a dosage of about 7 mg. In some embodiments, the DLL3 targeting trispecific protein is administered at a dosage of about 10 mg. In some embodiments, the DLL3 targeting trispecific protein is administered at a dosage of about 12 mg.In some embodiments, the DLL3 targeting trispecific protein is administered at a dosage of about 14 mg. In some embodiments, the DLL3 targeting trispecific protein is administered at a dosage of about 20 mg. In some embodiments, the DLL3 targeting trispecific protein is administered at a dosage of about 50 mg. In some embodiments, the DLL3 targeting trispecific protein is administered once a week. In some embodiments, the DLL3 targeting trispecific protein is administered twice a week. In some embodiments, the DLL3 targeting trispecific protein is administered once every two weeks. In some embodiments, the DLL3 targeting trispecific protein is administered once every three weeks. In some embodiments, the DLL3 targeting trispecific protein is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally.

[0004] Described herein is a method of treating cancer, comprising administering to a subject an effective amount of a DLL3-targeting trispecific protein, wherein the protein comprises (a) a first domain (A) that specifically binds to human CD3, (b) a second domain (B) that is a half-life extending domain, and (c) a third domain (C) that specifically binds to DLL3, wherein the domains are linked in the order of H2N-(A)-(B)-(C)-COOH or by linkers L1 and L2, and the DLL3-targeting trispecific protein is administered according to a schedule comprising the steps of: (i) administering a first dose of the DLL3-targeting trispecific protein; (ii) administering a second dose of the DLL3-targeting trispecific protein, the second dose being higher than the first dose. In some embodiments, the first dose is about 1 mg to about 100 mg. In some embodiments, the first dose is about 1 mg to about 50 mg. In some embodiments, the first dose is about 1 mg to about 20 mg. In some embodiments, the first dose is about 1 mg to about 10 mg. In some embodiments, the first dose is about 1 mg to about 5 mg. In some embodiments, the first dose is about 1 mg to about 3 mg. In some embodiments, the first dose is about 2000 μg. In some embodiments, the first dose is about 3600 μg. In some embodiments, the first dose is administered for about 1 week to about 36 weeks. In some embodiments, the first dose is administered for about 1 week to about 27 weeks. In some embodiments, the first dose is administered for about 1 week to about 18 weeks. In some embodiments, the first dose is administered for about 1 week to about 9 weeks. In some embodiments, the first dose is administered once a day. In some embodiments, the first dose is administered twice a day. In some embodiments, the first dose is administered three times a day. In some embodiments, the first dose is administered five times a day. In some embodiments, the first dose is administered once a week. In some embodiments, the first dose is administered twice a week. In some embodiments, the first dose is administered once every two weeks. In some embodiments, the first dose is administered once every three weeks.In some embodiments, the first dose is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the second dose is about 1 mg to about 100 mg. In some embodiments, the second dose is about 1 mg to about 50 mg. In some embodiments, the second dose is about 50 mg to about 100 mg. In some embodiments, the second dose is about 7.2 mg. In some embodiments, the second dose is about 12 mg. In some embodiments, the second dose is about 24 mg. In some embodiments, the second dose is about 36 mg. In some embodiments, the second dose is administered for about 1 week to about 36 weeks. In some embodiments, the second dose is administered for about 1 week to about 27 weeks. In some embodiments, the second dose is administered for about 1 week to about 18 weeks. In some embodiments, the second dose is administered for about 1 week to about 9 weeks. In some embodiments, the second dose is administered once a day. In some embodiments, the second dose is administered twice a day. In some embodiments, the second dose is administered three times a day. In some embodiments, the second dose is administered five times a day. In some embodiments, the second dose is administered once a week. In some embodiments, the second dose is administered twice a week. In some embodiments, the second dose is administered once every two weeks. In some embodiments, the second dose is administered once every three weeks. In some embodiments, the second dose is maintained until the end of the schedule after administration of the first dose. In some embodiments, the second dose is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally.

[0005] In some embodiments, the DLL3-targeting trispecific protein has an elimination half-life of at least 12 hours, at least 20 hours, at least 25 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, or at least 100 hours. In some embodiments, the third domain comprises a VHH domain. In some embodiments, the VHH domain is human, humanized, affinity matured, or a combination thereof. In some embodiments, the third domain comprises one or more sequences selected from the group consisting of SEQ ID NOs: 1-442. In some embodiments, the first domain comprises a variable light chain and a variable heavy chain, each capable of specifically binding to human CD3. In some embodiments, the first domain is humanized or human. In some embodiments, the second domain binds human serum albumin. In some embodiments, the second domain comprises an scFv, a variable heavy domain (VH), a variable light domain (VL), a peptide, a ligand, or a small molecule. In some embodiments, each of the linkers L1 and L2 is (GS) n (SEQ ID NO: 1809), (GGS) n (SEQ ID NO: 1810), (GGGS) n (SEQ ID NO: 1811), (GGSG) n (SEQ ID NO: 1812), (GGSGG) n (SEQ ID NO: 1813), (GGGGS) n(SEQ ID NO: 1814), or GGGGSGGGS (SEQ ID NO: 1808), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linkers L1 and L2 are each independently (GGGGS)4 (SEQ ID NO: 1817), (GGGGS)3 (SEQ ID NO: 1818), or GGGGSGGGS (SEQ ID NO: 1808). In some embodiments, the domains are linked in the order H2N-(C)-L1-(B)-L2-(A)-COOH. In some embodiments, the DLL3 targeting trispecific protein is less than about 80 kDa. In some embodiments, the DLL3 targeting trispecific protein is about 50 to about 75 kDa. In some embodiments, the DLL3 targeting trispecific protein is less than about 60 kDa. In some embodiments, the DLL3 targeting trispecific protein comprises a sequence selected from the group consisting of SEQ ID NOs: 1890 to 1891. In some embodiments, the DLL3 targeting trispecific protein comprises the sequence set forth in SEQ ID NO: 1890. In some embodiments, the cancer is a neoplastic disease, an autoimmune disease, or an infectious disease associated with DLL3. In some embodiments, the cancer is a neuroendocrine cancer, a prostate cancer, a lung cancer, a gastric cancer, a squamous cell carcinoma, a pancreatic cancer, a cholangiocarcinoma, a triple-negative breast cancer, or an ovarian cancer. In some embodiments, the cancer is a small cell lung cancer. In some embodiments, the cancer is a neuroendocrine prostate cancer.

[0006] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. [Brief description of the drawings]

[0007] The novel features of the invention are set forth with particularity in the appended claims. To better understand the features and advantages of the present invention, reference should be made to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized and the accompanying drawings in which: [Figure 1]Illustrates the various domains of an exemplary DLL3-targeting trispecific protein of the present disclosure. [Diagram 2] Illustrates the results of a T cell dependent cytotoxicity (TDCC) assay against DMS-153 cells using exemplary DLL3-targeting trispecific proteins containing the DLL3 binding domains of the present disclosure, DH18, DH11, DH67, and DH56. [Diagram 3] Illustrates the results of a TDCC assay on DMS-153 cells using an exemplary DLL3-targeting trispecific protein containing exemplary DLL3-binding domains of the disclosure, DH2, DH43, DH10, and DH6. [Figure 4] 1 illustrates the results of a TDCC assay on DMS-153 cells using exemplary DLL3-targeting trispecific proteins containing exemplary DLL3-binding domains of the disclosure, DH82, DH23, DH89, and DH17. [Diagram 5] Illustrates the results of a TDCC assay on DMS-153 cells using exemplary DLL3-targeting trispecific proteins containing exemplary DLL3-binding domains of the disclosure, DH83, DH12, DH61, and DH29. [Figure 6] Illustrates the results of a TDCC assay on DMS-153 cells using exemplary DLL3-targeting trispecific proteins containing exemplary DLL3 binding domains of the disclosure, DH58 and DH70, and a control trispecific protein. [Figure 7] 1 illustrates the results of a TDCC assay on DMS-153 cells using exemplary affinity matured DLL3-targeting trispecific proteins containing exemplary DLL3 targeting domains of the present disclosure, 1A011, 2E05, 1H012, 2E02, and 1C03. [Figure 8] 1 illustrates the results of a TDCC assay on DMS-153 cells using exemplary affinity matured DLL3-binding trispecific proteins containing exemplary DLL3 targeting domains of the present disclosure, 2E010, 2E01, 2H02, 2A04, and 2F11. [Figure 9] 1 illustrates the results of a TDCC assay on DMS-153 cells using exemplary affinity matured DLL3-binding trispecific proteins containing exemplary DLL3 targeting domains of the present disclosure, 2E011, 3C04, 4H04, 4H011, and 4D09. [Figure 10] Illustrates the results of a TDCC assay on DMS-153 cells using exemplary affinity matured DLL3-binding trispecific proteins containing exemplary DLL3 targeting domains of the disclosure, 4B07, 4E02, 4C06, 3H011, and 3D07. [Figure 11] Illustrates the results of a TDCC assay on DMS-153 cells using exemplary DLL3 binding domains 3H06 and 4B011 of the present disclosure, and exemplary affinity matured DLL3-targeted trispecific proteins containing parent DLL binder domains DH43, DH6, as well as a control trispecific protein. [Figure 12] Illustrates the results of a TDCC assay on DMS-153 cells using exemplary purified affinity matured CHO-expressed DLL3-binding trispecific proteins containing exemplary DLL3 targeting domains of the present disclosure, 2E05-M106Y, 2E05-M106Q, 4D09-M34L, and 4H11-M34L. [Figure 13] 13 illustrates the results of a TDCC assay on DMS-153 cells using an exemplary purified affinity matured CHO-expressed DLL3-targeted trispecific protein containing exemplary DLL3 binding domains of the present disclosure, 1A011 (labeled as 1A11 in FIG. 13), 1H012 (labeled as 1H12 in FIG. 13), 2E02, and 2E05. [Figure 14] Illustrates the results of a TDCC assay on DMS-153 cells using exemplary purified affinity matured CHO-expressed DLL3-targeted trispecific proteins containing exemplary DLL3 binding domains of the disclosure, 2H02, 3C04, 4D09, and 4H11. [Figure 15]Illustrates the results of a TDCC assay on DMS-153 cells using an exemplary purified DLL3-targeting trispecific protein containing exemplary DLL3-binding domains DH43 and DH6, and a control trispecific protein targeting GFP. [Figure 16] 1 illustrates the results of a TDCC assay on DMS-153 cells using an exemplary DLL3-targeting trispecific protein containing an exemplary DLL3-binding domain of the present disclosure from the second round of affinity maturation. [Figure 17] Illustrates an image of a 10-20% TRIS glycine SDS-PAGE loaded with 2.4 micrograms of non-reduced protein per lane and stained with Coomassie. Lane numbers are indicated by numbers at the top of the gel image, and migration of molecular weight standards is indicated by numbers to the right of the gel image (kilodaltons). Gel loading: lane 1 is empty, lane 2 is molecular weight standards, lane 3 is empty, lane 4 is anti-DLL3 trispecific containing DLL3 binding domain 51G2, lane 5 is anti-DLL3 trispecific containing DLL3 binding domain 51G10, lane 6 is anti-DLL3 trispecific containing DLL3 binding domain 51H5, lane 7 is anti-DLL3 trispecific containing DLL3 binding domain 51X5, lane 8 is anti-DLL3 trispecific containing DLL3 binding domain 52B1, lane 9 is anti-DLL3 trispecific containing DLL3 binding domain 52C4, lane 10 is anti-DLL3 trispecific containing DLL3 binding domain 52D4, lane 11 is anti-DLL3 trispecific containing DLL3 binding domain 51A2, lane 12 is anti-DLL3 trispecific containing DLL3 binding domain 51A5, lane 13 is anti-DLL3 trispecific containing DLL3 binding domain 51F3, lane 14 is empty, and lane 15 is empty. [Figure 18]Illustrated are the results of a TDCC assay on DMS-53 cells using exemplary purified affinity matured CHO-expressed DLL3-targeted trispecific proteins containing exemplary DLL3 binding domains of the present disclosure, 51G2, 51G10, 51H5, 51X5, 52B1, 52C4, 52D4, 51A2, and the parent DLL3 binder domain DH6, as well as a control trispecific protein. [Figure 19] Illustrates the results of a TDCC assay on DMS-153 cells using exemplary purified affinity matured CHO-expressed DLL3-targeted trispecific proteins of the disclosure containing exemplary DLL3 binding domains 51G2, 51G10, 51H5, 51X5, 52B1, 52C4, 52D4, 51A2, and the parent DLL3 binder domain DH6, as well as a control binding trispecific protein targeting GFP. [Figure 20] FIG. 1 provides a schematic diagram of a DLL3-targeting trispecific protein containing an exemplary DLL3 binding protein (DLL3 binder) of the present disclosure, a CD3 binding domain (anti-CD3 epsilon scFv), and an albumin binding (anti-ALB) domain in an anti-DLL3 anti-ALB:anti-CD3 orientation (TAC orientation). [Figure 21] FIG. 1 provides a schematic diagram of an exemplary DLL3-targeting trispecific protein of the present disclosure containing an exemplary DLL3 binding protein (DLL3 binder), a CD3 binding domain (anti-CD3 epsilon scFv), and an albumin binding (anti-ALB) domain in an anti-CD3:anti-ALB:anti-DLL3 orientation (CAT orientation). [Figure 22] Illustrated are the results of a T-cell dependent cytotoxicity (TDCC) assay against NCI-H2171 cells using an exemplary DLL3 trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, or an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) or bovine serum albumin (BSA). [Diagram 23]Illustrates the results of a T-cell dependent cytotoxicity (TDCC) assay against DMS-79 cells using an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, or an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence or absence of human serum albumin (HSA). [Figure 24] Illustrated are the results of a T-cell dependent cytotoxicity (TDCC) assay against SHP77 cells using an exemplary DLL3 trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, or an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) or bovine serum albumin (BSA). [Diagram 25] Illustrated are the results of a T-cell dependent cytotoxicity (TDCC) assay against WM2664 cells using an exemplary DLL3 trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, or an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) or bovine serum albumin (BSA). [Figure 26] 1 depicts binding of an exemplary DLL3 trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration to human T cells from four different donors compared to controls with secondary antibody only or cells with no antibody or trispecific molecule at all. [Figure 27] 1 depicts binding of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration to human T cells from four different donors compared to controls with secondary antibody only or cells with no antibody or trispecific molecule at all. [Figure 28]1 depicts binding of an exemplary DLL3-targeting trispecific protein containing a DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration to human DLL3-expressing cell lines, NCI-H82 (top left), SHP77 (top right), DMS53 (bottom left), or NCI-H2171 (bottom right), compared to a trispecific molecule with a GFP-binding domain. [Figure 29] 1 depicts binding of an exemplary DLL3-targeting trispecific protein containing the DLL3-binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration to human DLL3-expressing cell lines, NCI-H82 (top left), SHP77 (top right), DMS53 (bottom left), or NCI-H2171 (bottom right), compared to a trispecific molecule with a GFP-binding domain. [Diagram 30] Illustrates the results of a TDCC assay on NCI-H82 cells using an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Diagram 31] Illustrates the results of a TDCC assay on SHP77 cells using an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Diagram 32] Illustrates the results of a TDCC assay on DMS53 cells using an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Diagram 33]Illustrates the results of a TDCC assay on NCI-H2171 cells using an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Diagram 34] Illustrates the results of a TDCC assay on NCI-H82 cells using an exemplary DLL3-targeting trispecific protein containing the DLL3-binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Diagram 35] Illustrates the results of a TDCC assay on SHP77 cells using an exemplary DLL3-targeting trispecific protein containing the DLL3-binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Diagram 36] Illustrates the results of a TDCC assay on DMS53 cells using an exemplary DLL3-targeting trispecific protein containing the DLL3-binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Figure 37] Illustrates the results of a TDCC assay on NCI-H2171 cells using an exemplary DLL3-targeting trispecific protein containing the DLL3-binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Figure 38]Illustrates the results of flow cytometry measurements of CD69 expression on T cells co-incubated with NCI-H82 cells by titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Figure 39] Illustrates the results of flow cytometry measurements of CD25 expression on T cells co-incubated with NCI-H82 cells by titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Diagram 40] Illustrates the results of flow cytometry measurements of CD69 expression on T cells co-incubated with DMS53 cells by titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Diagram 41] Illustrates the results of flow cytometry measurements of CD25 expression on T cells co-incubated with DMS53 cells by titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA). [Diagram 42] Illustrates the results of flow cytometry measurements of CD69 expression on T cells co-incubated with NCI-H82 cells using T cells from four different donors, with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA). [Diagram 43] Illustrates the results of flow cytometry measurements of CD25 expression on T cells co-incubated with NCI-H82 cells by titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. [Diagram 44] Illustrates the results of flow cytometry measurements of CD69 expression on T cells co-incubated with DMS53 cells using T cells from four different donors, with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA). [Diagram 45] Illustrates the results of flow cytometry measurements of CD25 expression on T cells co-incubated with DMS53 cells by titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA). [Diagram 46] Illustrates the results of IFNγ measurements in conditioned medium from co-cultures of incubated T cells and NCI-H82 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA). [Figure 47] Illustrates the results of IFNγ measurements in conditioned medium from co-cultures of incubated T cells and SHP77 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA). [Figure 48] Illustrates the results of IL-2 measurement in conditioned medium from co-cultures of incubated T cells and NCI-H82 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA). [Figure 49] Illustrates the results of IL-2 measurement in conditioned medium from co-cultures of incubated T cells and SHP77 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA). [Figure 50] Illustrates the results of TNFα measurements in conditioned medium from co-cultures of incubated T cells and NCI-H82 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA). [Figure 51] Illustrates the results of TNFα measurements in conditioned medium from co-cultures of incubated T cells and SHP77 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA). [Figure 52] Illustrates the results of IFNγ measurements in conditioned medium from co-cultures of incubated T cells and NCI-H82 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA). [Figure 53]Illustrates the results of IFNγ measurements in conditioned medium from co-cultures of incubated T cells and SHP77 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA). [Figure 54] Illustrates the results of IL-2 measurement in conditioned medium from co-cultures of incubated T cells and NCI-H82 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA). [Figure 55] Illustrates the results of IL-2 measurement in conditioned medium from co-cultures of incubated T cells and SHP77 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA). [Figure 56] Illustrates the results of TNFα measurements in conditioned medium from co-cultures of incubated T cells and NCI-H82 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA). [Figure 57] Illustrates the results of TNFα measurements in conditioned medium from co-cultures of incubated T cells and SHP77 cells with titration of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA). [Figure 58]1 depicts that an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) or anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration was able to inhibit tumor growth in mice injected with a mixture of human T cells and NCI-H82 small cell lung cancer cells at doses of 20 μg / kg, 100 μg / kg, or 500 μg / kg. [Figure 59] 1 depicts an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration was able to eliminate NCI-H82 xenograft tumor growth in mice injected with human T cells at doses of 10 μg / kg and 100 μg / kg. [Figure 60] 1 depicts that an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration was able to inhibit tumor growth in mice injected with a mixture of human T cells and SHP77 small cell lung cancer cells at doses of 10 μg / kg and 100 μg / kg. [Figure 61] 1 depicts the pharmacokinetic profile of an exemplary DLL3-targeting trispecific protein containing the disclosed DLL3 binding domain, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration (ID numbers 1 and 2) or an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration (ID numbers 3 and 4). Plots show serum levels of DLL3-targeting trispecific protein at various time points after injection into cynomolgus monkeys at 0.3 mg / kg. [Figure 62] 1 depicts the pharmacokinetic profile of an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration at various time points after injection into cynomolgus monkeys at 1 mg / kg or 10 mg / kg. [Figure 63]1 depicts the transient cytokine increase following initial dosing of 1 mg / kg and 10 mg / kg of an exemplary DLL3-binding TriTAC molecule of the present disclosure or vehicle control. The top panel shows the transient increase in IFNγ, the second panel shows the transient increase in IL-6, and the third panel shows the transient increase in IL-10. [Figure 64] Illustrates the results of a TDCC assay on DMS53 cells using an exemplary DLL3-targeting trispecific protein containing the DLL3 binding domain of the present disclosure, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration using freshly thawed protein or using protein present in a cynomolgus monkey serum sample taken 168 hours after dosing with 10 mg / kg of the DLL3-targeting trispecific protein measured in the presence of 8.4% cynomolgus monkey serum. [Figure 65] Illustrates a DLL3 trispecific antigen binding protein Phase 1 / 2 trial design. [Figure 66] Patient treatment times, weekly doses per patient, number of previous treatments, and patient identification numbers will be documented. [Figure 67] Maximum percent target lesion response from baseline in each cohort is shown. [Figure 68] 1 shows the reduction of target lesions over time in patients. [Figure 69] Illustrates pharmacokinetic data of DLL3 trispecific antigen binding protein for different dosing cohorts. [Figure 70A] 13A-13D demonstrate flow analysis results. 13B-13D demonstrate T cell margination levels after treatment. [Figure 70B] 13A-13D demonstrate the results of flow analysis. 13B-13C demonstrate the induction of T cell activation markers following treatment. [Figure 71A] 13 demonstrates the change in target lesion diameter over time for patient 111. [Figure 71B] The CT scan illustrates a reduction in the sum of the target lesion diameters for patient 111. [Figure 72A]13 demonstrates the change in target lesion diameter over time for patient 112. [Fig. 72B] The CT scan illustrates a reduction in the sum of the target lesion diameters for patient 112. [Figure 73] 13 demonstrates the change in target lesion diameter over time for patient 113. [Fig. 74A] Concentration-time profiles by dose are shown. [Fig. 74B] Cmax by dose is shown. [Fig. 75A] Peripheral IL-6 concentrations are shown after the first dose and after repeat or target doses. [Fig. 75B] The concentration of MCP-1 is shown after the first dose and after repeat or target doses. [Fig. 75C] T cell margination (CD8+) is shown after the first dose and after repeat or target doses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In some embodiments, proteins that specifically bind to delta-like ligand 3 (DLL3), multispecific (e.g., trispecific) proteins containing the proteins, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making the proteins are described herein. Methods of using at least one of the disclosed DLL3 binding proteins, or DLL3-targeting trispecific proteins comprising the same, for the prevention and / or treatment of diseases, illnesses, and disorders are further provided. The DLL3-targeting trispecific protein has a half-life-extending domain, such as a domain that can specifically bind to DLL3, similar to CD3, and can specifically bind to human albumin (ALB). Figure 1 depicts one non-limiting example of a trispecific DLL3 binding protein. In some embodiments, the DLL3-targeting trispecific protein comprises an antibody, such as a trispecific antibody.

[0009] Specific Definitions An "antibody" typically refers to a Y-shaped tetrameric protein containing two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Human light chains contain a variable domain (VL) and a constant domain (CL), which can be readily classified as kappa or lambda based on amino acid sequence and gene locus. Each heavy chain contains one variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD, contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the classification of IgG, IgA, and IgD, the CH1 and CH2 domains are separated by a flexible hinge region, which is a proline- and cysteine-rich segment of variable length (usually about 10 to about 60 amino acids in IgG). The variable domains of both the light and heavy chains are joined to the constant domains by a "J" region of about 12 or more amino acids, with the heavy chains having a "D" region of about 10 additional amino acids. Each class of antibody contains interchain and intrachain disulfide bonds formed by paired cysteine ​​residues. There are two types of natural disulfide bridges or bonds in immunoglobulin molecules: interchain disulfide bonds and intrachain disulfide bonds. The location and number of interchain disulfide bonds vary with immunoglobulin class and species. Interchain disulfide bonds are located on the surface of the immunoglobulin, are accessible to solvent, and are usually relatively easy to reduce. In the human IgG1 isotype, there are four interchain disulfide bonds, one from each heavy chain to the light chain and two between heavy chains. Interchain disulfide bonds are not required for chain association. As is well known, the cysteine-rich IgG1 hinge region of the heavy chain is generally held to consist of three parts: the upper hinge, the core hinge, and the lower hinge. Those skilled in the art will understand that the IgG1 hinge region contains cysteines in the heavy chain that contain interchain disulfide bonds (two heavy / heavy, two heavy / light), which provide structural flexibility that facilitates Fab movement. The interchain disulfide bond between the light and heavy chains of IgG1 is formed between C214 of the kappa or lambda light chain and C220 in the upper hinge region of the heavy chain.The interchain disulfide bonds between the heavy chains are at positions C226 and C229 (all numbered according to the EU index according to Kabat, et al., below).

[0010] As used herein, the term "antibody" includes polyclonal antibodies, multiclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, polyvalent antibodies, anti-idiotypic antibodies, synthetic antibodies, immunospecific antibody fragments, e.g., Fd, Fab, F(ab')2, F(ab')2 fragments, single chain fragments (e.g., ScFv and ScFvFc), disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies (VH, VL, or VHH domains), such as sdAbs, including muteins and variants thereof, and any other immunoreactive molecule so long as it contains a domain that has a binding site for preferential association or binding with a DLL3 protein. Moreover, unless contextual constraints dictate otherwise, the term further includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The heavy-chain constant domains that correspond to the various classes of antibodies are typically designated by the corresponding lowercase Greek letters α, δ, ε, γ, and μ, respectively. The light chains of antibodies of any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domains.

[0011] In some embodiments, the DLL3 binding domain of the disclosed DLL3-targeting trispecific protein comprises a heavy chain-only antibody, such as a VH or VHH domain. In some instances, the DLL3-binding protein comprises a heavy chain-only antibody that is an engineered human VH domain. In some instances, the engineered human VH domain is generated by panning a phage display library. In some embodiments, the DLL3 binding domain of the disclosed DLL3-targeting trispecific protein comprises a VHH. The term "VHH" as used herein refers to a single-chain antibody binding domain that lacks a light chain. In some instances, the VHH is derived from a type of antibody found in camelids or cartilaginous fish that naturally lack light chains, or a synthetic non-immune VHH that can be constructed accordingly. Each heavy chain comprises a variable region encoded by V, D, and J exons. The VHH is optionally a naturally occurring VHH, e.g., a VHH from a camelid, or a recombinant protein that comprises a heavy chain variable domain. In some embodiments, the VHH is derived from a species selected from the group consisting of camel, llama, vicuña, guanaco, and cartilaginous fish (such as, but not limited to, shark). In another embodiment, the VHH is derived from an alpaca (such as, but not limited to, Huacaya Alpaca and Suri alpaca).

[0012] As used herein, "variable region" or "variable domain" refers to the fact that certain portions of the variable domain vary widely in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed throughout the variable domain of an antibody. It is concentrated in three segments called the complementarity determining regions (CDRs) or hypervariable regions in both the light chain (VL) and heavy chain (VH) variable domains. The more highly conserved portions of the variable domain are called the framework (FR). Native heavy and light chain variable domains each contain four FR regions that largely adopt a β-sheet configuration and are connected by three CDRs, forming loop junctions and, in some cases, forming part of the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular toxicity. In some cases, ScFv (for variable single-chain fragment) fragments obtained by genetic engineering associate in a single polypeptide chain the VH and VL domains of an antibody separated by a peptide linker.

[0013] In some embodiments of the present disclosure, the DLL3 binding domain, such as the DLL3 binding domain of the DLL3 targeting trispecific protein, comprises a single domain antibody, such as a heavy chain only antibody, such as a VH or VHH domain, and comprises three CDRs. Such heavy chain only antibodies, in some embodiments, bind DLL3 as a monomer that does not depend on dimerization with the VL (variable light chain) region for optimal binding affinity. In some embodiments of the present disclosure, the CD3 binding domain of the DLL3 targeting trispecific protein comprises an scFv. In some embodiments of the present disclosure, the albumin binding domain of the DLL3 targeting trispecific protein comprises a heavy chain only antibody, such as a single domain antibody comprising a VH or VHH domain.

[0014] The assignment of amino acids to each domain, framework region and CDR in some embodiments follows one of the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Ed.), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3rd Ed., Wily-VCH Verlag GmbH and Co or AbM (Oxford Molecular / MSI Pharmacopia), unless otherwise indicated. It is not intended that the CDRs of this disclosure necessarily correspond to the Kabat numbering convention. In some embodiments of the present disclosure, the DLL3 binding protein comprises a single domain antibody, such as a heavy chain only antibody, such as a VH or VHH domain, and comprises three CDRs. Such heavy chain only antibodies, in some embodiments, bind DLL3 as a monomer that does not depend on dimerization with the VL (variable light chain) region for optimal binding affinity.

[0015] "Kabat-like variable domain residue numbering" or "Kabat-like amino acid position numbering" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to omissions from, or insertions into, the FRs or CDRs of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence in regions of homology with the "standard" Kabat numbered sequence.

[0016] The term "framework" or "FR" residues (or regions) refers to variable domain residues other than the CDR or hypervariable region residues as defined herein. A "human consensus framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences.

[0017] As used herein, the term "percent (%) amino acid sequence identity" for a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

[0018] As used herein, "elimination half-life" is used in its ordinary sense as described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass a quantitative measure of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first-order kinetics) because the drug concentration does not usually reach the concentration required for saturation of the elimination process. The rate of an exponential process can be expressed by its rate constant k, which represents the fractional rate of change per unit of time, or by its half-life t1 / 2, which is the time required for 50% of the process to be completed. The units of these two constants are time-1 and time, respectively. The first-order rate constant and the half-life of a reaction are simply related (k×t1 / 2=0.693) and can be interchanged accordingly. First-order elimination kinetics dictates that a constant fraction of the drug is lost per unit of time, so that a plot of the logarithm of drug concentration versus time is linear for all times after the initial distribution phase (i.e., after drug absorption and distribution are complete). Drug elimination half-life can be accurately determined from such a graph.

[0019] As used herein, the term "binding affinity" refers to the affinity of a protein described in this disclosure for a binding target, and is expressed numerically using a "Kd" value. When two or more proteins are shown to have comparable binding affinities for their binding targets, the Kd values ​​for the binding of each protein to the binding targets are within ±2-fold of each other. When two or more proteins are shown to have comparable binding affinities for a single binding target, the Kd values ​​for the binding of each protein to the single binding target are within ±2-fold of each other. When a protein is shown to bind to two or more targets with comparable binding affinities, the Kd values ​​for the binding of the protein to the two or more targets are within ±2-fold of each other. Generally, a higher Kd value corresponds to weaker binding. In some embodiments, "Kd" is measured by radiolabeled antigen binding assay (RIA) or surface plasmon resonance assay using BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). In certain embodiments, the "on-rate" or "rate of association or association rate" or "k" and the "off-rate" or "rate of dissociation or dissociation rate" or "k" are also determined with surface plasmon resonance technology using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). In further embodiments, "Kd", "k" and "k" are measured using OCTET® Systems (Pall Life Sciences).In an exemplary method of measuring binding affinity using OCTET® Systems, a ligand, e.g., biotinylated human or cynomolgus DLL3, is immobilized on the OCTET® streptavidin capillary sensor tip surface, and then the streptavidin tip is activated with about 20-50 μg / ml of human or cynomolgus DLL3 protein according to the manufacturer's instructions. A solution of PBS / casein is also introduced as a blocker. For association kinetics measurements, DLL3 binding protein variants are introduced at concentrations ranging from about 10 ng / mL to about 100 μg / mL, about 50 ng / mL to about 5 μg / mL, or about 2 ng / mL to about 20 μg / mL. In some embodiments, DLL3 binding single domain proteins are used at concentrations ranging from about 2 ng / mL to about 20 μg / mL. Complete dissociation is observed in the case of negative control assay buffer without binding protein. The kinetic parameters of the binding reaction are then determined using an appropriate tool, for example ForteBio software.

[0020] One embodiment provides a DLL3 binding protein (also referred to herein as a DLL3 binding domain, such as the DLL3 binding domain of the DLL3 trispecific antibody of the present disclosure), which comprises a single domain antibody comprising a CDR1 sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 443-884 and 1887, a CDR2 sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 885-1326 and 1888, and a CDR3 sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 1327-1768 and 1889. In some embodiments, it is contemplated that the DLL3 binding protein of the present disclosure is fairly small, in some embodiments, 25 kD or less, 20 kD or less, 15 kDa or less, or 10 kDa or less. In certain examples, the EGFR binding protein, when a peptide or small molecule entity, is 5 kDa or less.

[0021] In one embodiment, the DLL3 targeting trispecific protein (also referred to herein as DLL3 binding trispecific protein, DLL3 trispecific protein, or DLL3 TriTAC™) comprises (a) a first domain (A) that specifically binds to human CD3, (b) a second domain (B) that is a half-life extension domain, and (c) a third domain (C) that specifically binds to DLL3. The three domains of the DLL3 targeting trispecific protein may be arranged in any order. Thus, it is contemplated that the domain order of the DLL3 targeting trispecific protein is as follows: H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, HN-(C)-(B)-(A)-COOH, or H2N-(C)-(A)-(B)-COOH.

[0022] In some embodiments, the DLL3 targeting trispecific protein has a domain order of H2N-(A)-(B)-(C)-COOH. In some embodiments, the DLL3 targeting trispecific protein has a domain order of H2N-(A)-(C)-(B)-COOH. In some embodiments, the DLL3 targeting trispecific protein has a domain order of H2N-(B)-(A)-(C)-COOH. In some embodiments, the DLL3 targeting trispecific protein has a domain order of H2N-(B)-(C)-(A)-COOH. In some embodiments, the DLL3 targeting trispecific protein has a domain order of H2N-(C)-(B)-(A)-COOH. In some embodiments, the DLL3 targeting trispecific protein has a domain order of H2N-(C)-(B)-(A)-COOH. In some embodiments, the DLL3 targeting trispecific protein has a domain order of H2N-(C)-(A)-(B)-COOH.

[0023] In some embodiments, the DLL3-targeting trispecific protein has an HSA (also referred to herein as ALB) binding domain as the central domain, such that the domain order is H2N-(A)-(B)-(C)-COOH, or H2N-(C)-(B)-(A)-COOH. In such embodiments in which the HSA binding domain is the central domain, it is contemplated that the CD3 and DLL3 binding domains are afforded additional flexibility for binding to their respective targets.

[0024] In some embodiments, the trispecific binding protein comprises a third domain that specifically binds to DLL3, where the third domain is optionally a DLL3-binding single domain antibody that binds to DLL3 with an affinity equal to or greater than that of a reference DLL3-binding parent molecule. The third domain, in some embodiments, comprises an affinity matured DLL3-binding molecule (e.g., an affinity matured DLL3-binding single domain antibody) and is derived from a DLL3-binding parent molecule that includes one or more amino acid mutations (e.g., stabilizing mutations, destabilizing mutations) relative to the DLL3-binding parent molecule. In some embodiments, the affinity matured DLL3-binding molecule has superior stability against a selected destabilizing agent compared to the reference DLL3-binding parent molecule. In some embodiments, the affinity matured DLL3-binding molecule is identified in a process that includes panning one or more pre-candidate DLL3-binding molecules derived from one or more DLL3-binding parent molecules expressed in a phage display library against a DLL3 protein, such as a human DLL3 protein. The pre-candidate DLL3-binding molecule, in some embodiments, includes amino acid substitutions in variable regions, CDRs, or framework residues relative to the parent molecule.

[0025] As used herein, "phage display" refers to a technique in which mutant polypeptides are displayed as fusion proteins to at least a portion of a coat protein on the surface of a phage, filamentous phage, or particle. The utility of phage display lies in the fact that a large library of randomized protein variants can be rapidly and efficiently selected for sequences that bind to a target molecule with high affinity. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides for those with specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins by fusing to either gene III or gene VIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct. Biol, 3:355-362 (1992), and references cited therein. In monovalent phage display, a protein or peptide library is fused to gene III or a portion thereof and expressed at low levels in the presence of wild-type gene III protein, such that the phage particle displays one copy of the fusion protein or none of the fusion protein. Because avidity effects are reduced compared to polyvalent phages, selection is based on affinity for endogenous ligands and phagemid vectors are used, which facilitate DNA manipulation. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).

[0026] In some embodiments, the panning comprises using varying binding times and concentrations to identify DLL3-binding molecules with increased or decreased on-rates from the pre-candidate DLL3-binding molecules. In some embodiments, the panning comprises using varying wash times to identify DLL3-binding molecules with increased or decreased on-rates from the pre-candidate DLL3-binding molecules. In some embodiments, the panning comprises using varying binding times and varying wash times. In some embodiments, one or more stabilizing mutations are combined to increase the stability of the affinity-matured DLL3-binding molecules, for example, by shuffling to generate a second-stage combinatorial library from such mutants and performing a second round of panning followed by binding selection.

[0027] In some embodiments, the affinity matured DLL3 binding molecule has an affinity for DLL3 protein (such as human DLL3 protein) that is equal to or greater than the DLL3-binding parent molecule, but has reduced or, in some embodiments, increased cross-reactivity with selected substances, such as ligands, proteins, antigens, etc., other than the DLL3 epitope for which the DLL3-binding parent molecule is specific or designed to be specific. Regarding the latter, in some embodiments, the affinity matured DLL3 binding molecule is more successful in animal model testing when it is reacted with both human DLL3 and the corresponding target of the animal model, mouse DLL3 or cynomolgus DLL3. In some embodiments, the parent DLL3 binding molecule binds to human DLL3 with an affinity of about 10 nM or less and binds to cynomolgus DLL3 with an affinity of about 15 nM or less. In some embodiments, the affinity matured DLL3 binding molecule identified after one round of panning binds to human DLL3 with an affinity of about 5 nM or less and binds to cynomolgus DLL3 with an affinity of about 7.5 nM or less. In some embodiments, affinity matured DLL3 binding molecules identified after two rounds of panning bind to human DLL3 with an affinity of about 2.5 nM or less and bind to cynomolgus DLL3 with an affinity of about 3.5 nM or less.

[0028] In some embodiments, Domain A, Domain B, and Domain C of the trispecific binding proteins of the present disclosure are antigen-specific binding domain polypeptides that independently bind to targets, such as targets on disease cells, or targets on other cells that support a disease state, such as targets on stromal cells that support tumor growth or targets on immune cells that support disease-mediated immune suppression. In some examples, the antigen-specific binding domains include antibodies, heavy chain only antibodies, including single chain antibodies, Fabs, Fvs, T cell receptor binding domains, ligand binding domains, receptor binding domains, domain antibodies, single domain antibodies, minibodies, nanobodies, peptibodies, or various other antibody mimetics, such as affimers, affitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, Kunitz domain-based proteins, avimers, knottins, finomers, darpins, affibodies, affilins, monobodies, and armadillo repeat protein-based proteins.

[0029] In some embodiments, the DLL3-targeting trispecific proteins described herein comprise a DLL3-binding polypeptide having a sequence selected from SEQ ID NOs: 1-442 and 1886, subsequences thereof, and variants thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3-binding polypeptide (i.e., a third domain (C)) having at least 70%-95% or more homology to a sequence selected from SEQ ID NOs: 1-442 and 1886, subsequences thereof, and variants thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3-binding polypeptide (i.e., a third domain (C)) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to a sequence selected from the group consisting of SEQ ID NOs: 1-442 and 1886, subsequences thereof, and variants thereof. In some embodiments, the trispecific antigen binding protein comprises a DLL3 binding polypeptide (i.e., a third domain (C)) having at least 70%-95% or more identity to a sequence selected from SEQ ID NOs: 1-442 and 1886, subsequences thereof, and variants thereof. In some embodiments, the trispecific antigen binding protein comprises a DLL3 binding polypeptide (i.e., a third domain (C)) having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a sequence selected from the group consisting of SEQ ID NOs: 1-442 and 1886, subsequences thereof, and variants thereof.

[0030] The DLL3-targeting trispecific proteins described herein are designed to be able to specifically target cells expressing DLL3 by recruiting cytotoxic T cells. In some embodiments, this improves efficacy compared to ADCC (antibody-dependent cellular cytotoxicity), which uses full-length antibodies directed against a single antigen and cannot directly recruit cytotoxic T cells. In contrast, by engaging CD3 molecules specifically expressed on these cells, DLL3-targeting trispecific proteins can crosslink cytotoxic T cells with cells expressing DLL3 in a highly specific manner, thereby directing the cytotoxic potential of T cells to the target cells. The DLL3-targeting trispecific proteins described herein bind to cytotoxic T cells by binding to surface-expressed CD3 protein, which forms part of the TCR. The simultaneous binding of multiple DLL3 trispecific antigen-binding proteins to CD3 and DLL3 expressed on the surface of a particular cell causes T cell activation and mediates the subsequent lysis of the particular DLL3-expressing cell. Therefore, the DLL3 targeting trispecific protein is intended to display strong, specific and efficient target cell killing.In some embodiments, the DLL3 targeting trispecific protein described herein stimulates the killing of target cells by cytotoxic T cells to eliminate pathogenic cells (e.g., tumor cells expressing DLL3).In some such embodiments, cells are selectively eliminated, thereby reducing the possibility of toxic side effects.

[0031] The DLL3-targeting trispecific proteins described herein further confer advantages over conventional monoclonal antibodies and other smaller bispecific molecules. In general, the efficacy of recombinant protein pharmaceuticals is highly dependent on the intrinsic pharmacokinetics of the protein itself. One advantage here is that the DLL3-targeting trispecific proteins described herein have an extended pharmacokinetic elimination half-life by having a half-life extension domain, such as a domain that specifically binds to serum albumin protein (e.g., human serum albumin protein, HSA). In this regard, the DLL3-targeting trispecific proteins described herein have an extended serum elimination half-life of about 2, 3, about 5, about 7, about 10, or about 14 days, depending on the embodiment. This is in contrast to other binding proteins, such as BiTE or DART molecules, which have relatively very short elimination half-lives. For example, the CD19×CD3 bispecific scFv-scFv fusion molecules of BiTEs require drug delivery by continuous infusion (intravenous) due to their short elimination half-life. The longer intrinsic half-life of the DLL3-targeting trispecific proteins solves this problem, thereby increasing therapeutic possibilities such as lower dose pharmaceutical formulations, reduced regular dosing, and / or novel pharmaceutical compositions.

[0032] The DLL3 targeting trispecific proteins described herein further have an optimal size for enhanced tissue penetration and tissue distribution. Larger sizes limit or prevent the penetration or distribution of the protein in the target tissue. The DLL3 targeting trispecific proteins described herein avoid this by having a small size that enhances tissue penetration and distribution. Accordingly, the DLL3 targeting trispecific proteins described herein, in some embodiments, have a size of about 50 kDa to about 80 kDa, about 50 kDa to about 75 kDa, about 50 kDa to about 70 kDa, or about 50 kDa to about 65 kDa. In some embodiments, the size of the DLL3 targeting trispecific proteins is less than about 60 kDa. Thus, the size of the DLL3 targeting trispecific proteins is more advantageous than IgG antibodies, which are about 150 kDa, and than BiTE and DART bispecific antibody molecules, which are about 55 kDa but do not have extended half-lives and are therefore quickly cleared by the kidney.

[0033] In further embodiments, the DLL3 targeting trispecific proteins described herein have an optimal size for enhanced tissue penetration and distribution. In these embodiments, the DLL3 targeting trispecific proteins are constructed to be as small as possible while retaining specificity for their targets. Accordingly, in these embodiments, the DLL3 targeting trispecific proteins described herein have a size of about 20 kDa to about 40 kDa, or about 25 kDa to about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, or about 65 kDa. In some embodiments, the DLL3-targeting trispecific proteins described herein have a size of about 50 kDa, 49 kDa, 48 kDa, 47 kDa, 46 kDa, 45 kDa, 44 kDa, 43 kDa, 42 kDa, 41 kDa, 40 kDa, about 39 kDa, about 38 kDa, about 37 kDa, about 36 kDa, about 35 kDa, about 34 kDa, about 33 kDa, about 32 kDa, about 31 kDa, about 30 kDa, about 29 kDa, about 28 kDa, about 27 kDa, about 26 kDa, about 25 kDa, about 24 kDa, about 23 kDa, about 22 kDa, about 21 kDa, or about 20 kDa. A typical approach to small size is by using single domain antibody (sdAb) fragments for each of the domains. For example, a particular DLL3 trispecific antigen binding protein has an anti-CD3 sdAb, an anti-ALB sdAb, and an sdAb for DLL3. This reduces the size of a typical DLL3 trispecific antigen binding protein to less than 60 kDa. Thus, in some embodiments, the domains of a DLL3 targeting trispecific protein are all single domain antibody (sdAb) fragments. In some embodiments, it is contemplated that the DLL3 binding protein is fairly small, less than 25 kDa, less than 20 kDa, less than 15 kDa, or less than 10 kDa in some embodiments. In certain examples, the DLL3 binding protein is less than 5 kDa when it is a peptide or small molecule entity.

[0034] In other embodiments, the DLL3-targeting trispecific proteins described herein include small molecule entity (SME) binders for ALB, DLL3, CD3, or all of the above. The SME binders are small molecules, averaging about 500-2000 Da in size, that are attached to the DLL3-targeting trispecific proteins by known methods, such as sortase ligation or conjugation. In these examples, one of the domains of the DLL3-targeting trispecific antigen binding protein is a sortase recognition sequence, LPETG (SEQ ID NO: 1896). To bind the SME binder to the DLL3-targeting trispecific antigen binding protein having a sortase recognition sequence, the protein is incubated with sortase and the SME binder, whereby the sortase binds the SME binder to the recognition sequence. In yet other embodiments, the DLL3-binding domain of the DLL3-targeting trispecific proteins described herein includes a knottin peptide for binding to DLL3. Knottins are disulfide-stable peptides with a cysteine ​​knot scaffold and have an average size of about 3.5 kDa.Knottins have been proposed for binding to specific tumor molecules, such as DLL3.In further embodiments, the third domain that binds to DLL3 of the DLL3-targeting trispecific protein described herein comprises a natural DLL3 ligand.

[0035] Another feature of the DLL3 targeting trispecific proteins described herein is that they are single polypeptide designs with flexible domain connections. This allows for easy production and manufacturing of DLL3 targeting trispecific proteins, because they can be encoded by a single cDNA molecule that can be easily incorporated into a vector. Furthermore, because the DLL3 targeting trispecific proteins described herein are monomeric single polypeptide chains, there is no problem with chain pairing and no requirement for dimerization. It is contemplated that the DLL3 targeting trispecific proteins described herein have a reduced tendency to aggregate, unlike other reported molecules, such as bispecific proteins with Fcγ immunoglobulin domains.

[0036] In the DLL3-targeting trispecific proteins described herein, the domains are linked, in some embodiments, by internal linkers L1 and L2, where L1 links the first and second domains of the DLL3-targeting trispecific protein, and L2 links the second and third domains of the DLL3-targeting trispecific protein. The linkers L1 and L2 have an optimized length and / or amino acid composition. In some embodiments, the linkers L1 and L2 have the same length and amino acid composition. In other embodiments, L1 and L2 are different. In certain embodiments, the internal linkers L1 and / or L2 are "short", i.e., comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the internal linker comprises about 12 amino acid residues or less. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the internal linkers L1 and / or L2 are "long", i.e., comprise 15, 20, or 25 amino acid residues. In some embodiments, these internal linkers comprise from about 3 to about 15, e.g., 8, 9, or 10, consecutive amino acid residues. With regard to the amino acid composition of the internal linkers L1 and L2, peptides are selected for properties that provide flexibility to the DLL3-targeting trispecific protein, do not interfere with the binding domain, and are also not resistant to cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. Examples of suitable internal linkers for linking domains in a DLL3-targeting trispecific protein include, but are not limited to, (GS) n (SEQ ID NO: 1809), (GGS) n (SEQ ID NO: 1810), (GGGS) n (SEQ ID NO: 1811), (GGSG) n (SEQ ID NO: 1812), (GGSGG) n (SEQ ID NO: 1813), (GGGGS) n (SEQ ID NO: 1814), (GGGG) n (SEQ ID NO: 1815), or (GGG) n(SEQ ID NO: 1816), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the internal linker L1 and / or L2 is (GGGGS)4 (SEQ ID NO: 1817) or (GGGGS)3 (SEQ ID NO: 1818). In another embodiment, the internal linker L1 and / or L2 is GGGGSGGGS (SEQ ID NO: 1808).

[0037] In some cases, domains within the DLL3-targeting trispecific proteins are conjugated using enzymatic site-specific conjugation methods, including the use of mammalian or bacterial transglutaminase enzymes. Microbial transglutaminase (mTG) is a versatile tool in modern research and biotechnology. The availability of relatively pure enzyme in large quantities, its ease of use, and lack of regulation by calcium or guanosine-5'-triphosphate (GTP) have made mTG the primary cross-linking enzyme used in the food industry and biotechnology fields. Currently, mTG is used in many applications to conjugate proteins and peptides to small molecules, polymers, surfaces, DNA and other proteins. See Pavel Strp, Veracity of microbial transglutaminase, Bioconjugate Chem. 25, 5, 855-862).

[0038] In some examples, a DLL3-targeting trispecific protein is provided, in which one of the domains contains an acceptor glutamine in the constant region that can be conjugated to another domain via a lysine-based linker (e.g., any primary amine chain that is a substrate for TGase, including alkylamines, oxoamines), where conjugation occurs only on one or more acceptor glutamine residues present in the targeting portion outside the antigen binding site (e.g., outside the variable region, within the constant region). Thus, conjugation does not occur on glutamines within the variable region, i.e., glutamines that are (at least partially) surface exposed. The trispecific protein is formed, in some examples, by reacting one of the domains with a lysine-based linker in the presence of TGase.

[0039] In some embodiments, when one or more domains in the DLL3 targeting triple binding protein are directly linked, a hybrid vector is made, and the DNAs encoding the directly linked domains are directly linked to each other.In some embodiments, when a linker is used, the DNA encoding the first domain of the three domains is ligated to the DNA encoding one end of the first linker portion, the DNA encoding the second domain of the three domains is ligated to the other end of the first linker portion, and the DNA encoding the second domain of the three domains is ligated to one end of the second linker portion, and the DNA encoding the third domain of the three domains is ligated to the other end of the second linker portion, where the first domain, the second domain, and the third domain are separate, and where the first domain, the second domain, and the third domain are independently selected from domain A, domain B, and domain C.Such ligation is carried out, for example, consecutively or as a three-way ligation.

[0040] CD3-binding domain The specificity of a T cell response is mediated by the recognition of antigen (displayed in the context of the major histocompatibility complex, MHC) by the TCR. As part of the TCR, CD3 is a protein complex present on the cell surface that includes the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. To comprise a complete TCR, CD3 binds together with the α (alpha) and β (beta) chains of the TCR, as well as CD3ζ (zeta). Clustering of CD3 on the T cell, such as by immobilized anti-CD3 antibodies, triggers T cell activation that resembles T cell receptor engagement but is independent of the specificity typical of that clone.

[0041] In one aspect, the DLL3-targeting trispecific proteins described herein comprise a domain that specifically binds to CD3. In one aspect, the DLL3-targeting trispecific proteins described herein comprise a domain that specifically binds to human CD3. In some embodiments, the DLL3-targeting trispecific proteins described herein comprise a domain that specifically binds to CD3γ. In some embodiments, the DLL3-targeting trispecific proteins described herein comprise a domain that specifically binds to CD3δ. In some embodiments, the DLL3-targeting trispecific proteins described herein comprise a domain that specifically binds to CD3ε.

[0042] In further embodiments, the DLL3 targeting trispecific proteins described herein comprise a domain that specifically binds to TCR. In certain examples, the DLL3 targeting trispecific proteins described herein comprise a domain that specifically binds the alpha chain of TCR. In certain examples, the DLL3 targeting trispecific proteins described herein comprise a domain that specifically binds the beta chain of TCR.

[0043] In certain embodiments, the CD3 binding domains of the DLL3-targeting trispecific proteins described herein not only exhibit potent CD3-binding affinity with human CD3, but also exhibit excellent cross-reactivity with the respective cynomolgus monkey CD3 proteins.

[0044] In some embodiments, the CD3 binding domain of the DLL3 trispecific antigen binding protein can be any domain that binds to CD3, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies.In some instances, it is beneficial for the CD3 binding domain to be derived from the same species that the DLL3 trispecific antigen binding protein will ultimately be used in.For example, for human use, it may be beneficial for the CD3 binding domain of the DLL3 trispecific antigen binding protein to include human or humanized residues from the antigen binding domain of an antibody or antibody fragment.

[0045] Thus, in one aspect, the antigen binding domain comprises a humanized or human antibody or antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-CD3 binding domain comprises a light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of one or more (e.g., all three) of the humanized or human anti-CD3 binding domains described herein, and / or a heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of one or more (e.g., all three) of the humanized or human anti-CD3 binding domains described herein, including one or more, all three LC CDRs and one or more, all three HC CDRs.

[0046] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human light chain variable region specific for CD3, the light chain variable region specific for CD3 comprises human or non-human light chain CDRs in a human light chain framework region. In some examples, the light chain framework region is a λ (lambda) light chain framework. In other examples, the light chain framework region is a κ (kappa) light chain framework.

[0047] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human heavy chain variable region specific for CD3, where the heavy chain variable region specific for CD3 comprises human or non-human heavy chain CDRs in a human heavy chain framework region.

[0048] In certain examples, the complementarity determining regions of the heavy and / or light chains are selected from, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66, or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12. 5, derived from known anti-CD3 antibodies such as F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, and WT-31.

[0049] In one embodiment, the anti-CD3 binding domain is a single chain variable fragment (scFv) comprising a light chain and a heavy chain of the amino acid sequences provided herein. As used herein, a "single chain variable fragment" or "scFv" refers to an antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the variable regions of the light and heavy chains are contiguously linked by a short, flexible polypeptide linker, expressible as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. In embodiments, the anti-CD3 binding domain comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of a light chain variable region provided herein, or a sequence having 95-99% identity to an amino acid sequence provided herein; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of a heavy chain variable region provided herein, or a sequence having 95-99% identity to an amino acid sequence provided herein. In some examples, the anti-CD3 binding domain comprises a sequence selected from SEQ ID NOs: 1793-1807, or a sequence that is at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a sequence selected from SEQ ID NOs: 1793-1807. In some examples, the anti-CD3 binding domain comprises three heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3) and three light chain CDRs. The heavy chain CDR1 (HC CDR1) of the CD3 binding domain comprises a sequence selected from SEQ ID NOs: 1820-1831, or a sequence that contains one or more modifications or substitutions in a sequence selected from SEQ ID NOs: 1820-1831, or is at least about 80% to about 99% identical.The heavy chain CDR2 (HC CDR2) of the CD3 binding domain comprises a sequence selected from SEQ ID NOs: 1832 to 1841, or a sequence containing one or more modifications or substitutions in the sequence selected from SEQ ID NOs: 1832 to 1841. The heavy chain CDR3 (HC CDR3) of the CD3 binding domain comprises a sequence selected from SEQ ID NOs: 1842 to 1853, or a sequence containing one or more modifications or substitutions in the sequence selected from SEQ ID NOs: 1842 to 1853. The light chain CDR1 (LC CDR1) of the CD3 binding domain comprises a sequence selected from SEQ ID NOs: 1852 to 1864, or a sequence containing one or more modifications or substitutions in the sequence selected from SEQ ID NOs: 1852 to 1864. The light chain CDR2 (LC CDR2) of the CD3 binding domain comprises a sequence selected from SEQ ID NOs: 1865 to 1877, or a sequence containing one or more modifications or substitutions in the sequence selected from SEQ ID NOs: 1865 to 1877. The light chain CDR3 (LC CDR3) of the CD3 binding domain comprises a sequence selected from SEQ ID NOs: 1878-1884, or a sequence containing one or more modifications or substitutions in a sequence selected from SEQ ID NOs: 1878-1884. In one embodiment, the humanized or human anti-CD3 binding domain is an scFv, and a light chain variable region comprising an amino acid sequence described herein is linked to a heavy chain variable region comprising an amino acid sequence described herein by an scFv linker. The light and heavy chain variable regions of the scFv can be in any of the following orientations: light chain variable region-scFv linker-heavy chain variable region, or heavy chain variable region-scFv linker-light chain variable region.

[0050] In some examples, scFvs that bind to CD3 are prepared according to known methods. For example, scFv molecules can be made by linking together VH and VL regions using a flexible polypeptide linker. The scFv molecules include an scFv linker (e.g., a Ser-Gly linker) with an optimized length and / or amino acid composition. Accordingly, in some embodiments, the length of the scFv linker is such that the VH or VL domain can bind intermolecularly with another variable domain to form a CD3 binding site. In certain embodiments, such scFv linkers are "short", i.e., consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the scFv linker consists of about 12 or less amino acid residues. In the case of 0 amino acid residues, the scFv linker is a peptide bond. In some embodiments, these scFv linkers consist of about 3 to about 15, e.g., 8, 9, or 10, consecutive amino acid residues. Regarding the amino acid composition of the scFv linker, a peptide is selected that provides flexibility and does not interfere with the variable domains, as well as allows interchain folding to join the two variable domains to form a functional CD3 binding site. For example, scFv linkers that contain glycine and serine residues generally provide protease resistance. In some embodiments, the linker in the scFv contains glycine and serine residues. The amino acid sequence of the scFv linker can be optimized, for example, by phage display methods to improve CD3 binding and production yield of the scFv. Examples of peptide scFv linkers suitable for linking the variable light and variable heavy domains in an scFv include (GS) n (SEQ ID NO: 1809), (GGS) n (SEQ ID NO: 1810), (GGGS) n (SEQ ID NO: 1811), (GGSG) n (SEQ ID NO: 1812), (GGSGG) n (SEQ ID NO: 1813), (GGGGS) n (SEQ ID NO: 1814), (GGGG) n (SEQ ID NO: 1815), or (GGG) n(SEQ ID NO: 1816), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the scFv linker can be (GGGGS)4 (SEQ ID NO: 1817), or (GGGGS)3 (SEQ ID NO: 1818). In some embodiments, the linker comprises a sequence made up of any combination of the linkers set forth in SEQ ID NOs: 1809-1818, the length of such linkers being, in some instances, up to 15 amino acids or longer. Variation in linker length may retain or enhance activity, resulting in superior efficacy in activity studies.

[0051] In some embodiments, the CD3 binding domain of a DLL3-targeting trispecific antigen binding protein has an affinity for CD3 on CD3-expressing cells with a KD of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In some embodiments, the CD3 binding domain of a DLL3-targeting trispecific antigen binding protein has an affinity for CD3 epsilon, gamma, or delta with a KD of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In a further embodiment, the CD3 binding domain of the DLL3-targeting trispecific antigen binding protein has a low affinity for CD3 (ie, about 100 nM or greater).

[0052] Affinity for binding to CD3 can be determined, for example, by the ability of the DLL3-targeting trispecific antigen binding protein itself or its CD3-binding domain to bind to CD3 coated on an assay plate, CD3 displayed on a microbial cell surface, CD3 in solution, etc. The binding activity of the DLL3-targeting trispecific antigen binding protein of the present disclosure, or its CD3-binding domain, to CD3 can be analyzed by immobilizing a ligand (e.g., CD3) or the DLL3-targeting trispecific antigen binding protein itself or its CD3-binding domain to beads, substrates, cells, etc. The agent can be added to the binding partner in an appropriate buffer and incubated for a period of time at a predetermined temperature. After washing to remove unbound material, the binding protein can be released, for example, with SDS, high pH buffer, etc., and analyzed, for example, by surface plasmon resonance (SPR).

[0053] Half-life extension domain Contemplated herein are domains that extend the half-life of an antigen-binding domain, including, but not limited to, albumin-binding domains, Fc domains, small molecules, and other half-life extending domains known in the art.

[0054] Human albumin (ALB) (molecular mass 67 kDa) is the most abundant protein in plasma, present at approximately 50 mg / ml (600 μM), with a half-life of approximately 20 days in humans. ALB helps maintain plasma pH, contributes to colloidal blood pressure, functions as a carrier for many metabolites and fatty acids, and serves as the major drug transport protein of plasma.

[0055] Non-covalent association with albumin extends the elimination half-life of short-lived proteins. For example, recombinant fusion of albumin-binding domain to Fab fragments resulted in 25-fold and 58-fold in vivo clearance and 26-fold and 37-fold half-life extensions when administered intravenously to mice and rabbits, respectively, compared to administration of the Fab fragment alone. In another example, when insulin was acylated with fatty acids to promote association with albumin, a delayed effect was observed when injected subcutaneously into rabbits or pigs. Collectively, such studies demonstrate the link between albumin binding and delayed action.

[0056] In one aspect, the DLL3-targeting trispecific protein described herein comprises a half-life extension domain, e.g., a domain that specifically binds to ALB. In some embodiments, the ALB-binding domain of the DLL3-targeting trispecific antigen-binding protein can be any domain that binds to ALB, including, but not limited to, a domain derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some embodiments, the ALB-binding domain is a single-chain variable fragment (scFv) specific for HSA, a single-domain antibody, e.g., a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of a single-domain antibody derived from camelid, a peptide, a ligand, or a small molecule entity. In certain embodiments, the ALB-binding domain is a single-domain antibody. In other embodiments, the HSA-binding domain is a peptide. In some embodiments, the HSA-binding domain is a small molecule. It is contemplated that the HSA binding domain of the DLL3 trispecific antigen binding protein is fairly small, in some embodiments, 25 kDa or less, 20 kDa or less, 15 kDa or less, or 10 kDa or less. In certain instances, the ALB binding domain is 5 kDa or less when it is a peptide or small molecule entity.

[0057] The half-life prolonging domain of the DLL3-targeting trispecific antigen binding protein results in changes in the pharmacokinetics and pharmacodynamics of the DLL3-targeting trispecific antigen binding protein itself. As described above, the half-life prolonging domain extends the elimination half-life. The half-life prolonging domain also alters the pharmacokinetic properties of the trispecific antigen binding protein, including changes in tissue distribution, penetration, and diffusion. In some embodiments, the half-life prolonging domain results in improved tissue (including tumor) targeting, tissue distribution, tissue penetration, diffusion within tissues, and enhanced efficacy compared to proteins without the half-life prolonging domain. In one embodiment, the method of treatment effectively and efficiently utilizes reduced amounts of the trispecific antigen binding protein, resulting in reduced side effects, such as reduced cytotoxicity in non-tumor cells.

[0058] Furthermore, the binding affinity of the half-life prolonging domain can be selected to target the specific elimination half-life of a particular trispecific antigen binding protein. Thus, in some embodiments, the half-life prolonging domain has a high binding affinity. In other embodiments, the half-life prolonging domain has a moderate binding affinity. In still other embodiments, the half-life prolonging domain has a low or marginal binding affinity. Exemplary binding affinities include KD concentrations of 10 nM or less (high), between 10 nM and 100 nM (medium), and above 100 nM (low). As described above, the binding affinity to ALB is determined by known methods, such as surface plasmon resonance (SPR). In some embodiments, the ALB binding domain described herein comprises a single domain antibody.

[0059] In some embodiments, the half-life extension domain comprises a sequence selected from SEQ ID NOs: 1769-1778, or a sequence at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1769-1778. In some examples, the half-life extension comprises three heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3) and three light chain CDRs. In some examples, the half-life extension comprises three heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3), or three light chain CDRs. The heavy chain CDR1 (HC CDR1) of the half-life prolonging domain, in some embodiments, comprises a sequence selected from SEQ ID NOs: 1782-1784, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID NOs: 1782-1784, or is at least about 80% to about 99%. The heavy chain CDR2 (HC CDR2) of the half-life prolonging domain, in some embodiments, comprises a sequence selected from SEQ ID NOs: 1785-1790, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID NOs: 1785-1790. The heavy chain CDR3 (HC CDR3) of the CD3 binding domain comprises a sequence selected from SEQ ID NOs: 1791 or 1792, or a sequence comprising one or more modifications or substitutions in 1792, SEQ ID NOs: 1791 or 1792.

[0060] DLL3-binding domain DLL3 (also known as delta-like ligand 3 or SCDO1) is a member of the delta-like family of Notch DSL ligands. Representative DLL3 protein orthologs include, but are not limited to, human (accession numbers NP_058637 and NP_982353), chimpanzee (accession number XP_003316395), mouse (accession number NP_031892), and rat (accession number NP_446118). In humans, the DLL3 gene consists of eight exons spanning 9.5 kbp located on chromosome 19q13. Alternative splicing within the last exon results in two processed transcripts, one of 2389 bases (accession number NM_016941) and one of 2052 bases (accession number NM_203486). The former transcript encodes a 618 amino acid protein (accession number NP_058637) and the latter encodes a 587 amino acid protein (accession number NP_982353). These two protein isoforms of DLL3 share an overall 100% identity across their extracellular and transmembrane domains, differing only in that the longer isoform contains an extended cytoplasmic tail containing 32 additional residues at the carboxy terminus of the protein. The extracellular region of the DLL3 protein contains six EGF-like domains, a single DSL domain, and an N-terminal domain. The EGF domains are generally recognized to occur approximately at amino acid residues 216-249 (domain 1), 274-310 (domain 2), 312-351 (domain 3), 353-389 (domain 4), 391-427 (domain 5), and 429-465 (domain 6), with the DSL domain occurring approximately at amino acid residues 176-215 of hDLL3 and the N-terminal domain occurring approximately at amino acid residues 27-175 of hDLL3. Each of the EGF-like domains, the DSL domain, and the N-terminal domain comprise portions of the DLL3 protein that are defined by distinct amino acid sequences. The EGF-like domains are, in some embodiments, referred to as EGF1-EGF6, with EGF1 being closest to the N-terminal portion of the protein.In general, DSL ligands are composed of the following series of structural domains: a unique N-terminal domain, followed by a conserved DSL domain, multiple tandem epidermal growth factor (EGF)-like repeats, a transmembrane domain, and a cytoplasmic domain that is not highly conserved across ligands but contains multiple lysine residues that are potential sites for ubiquitination by a unique E3 ubiquitin ligase. The DSL domain is a degenerate EGF domain that is necessary but not sufficient for interaction with the Notch receptor. In addition, the first two EGF-like repeats of most DSL ligands contain a smaller protein sequence motif known as a DOS domain that cooperatively interacts with the DSL domain in activating Notch signaling.

[0061] In some embodiments, the disclosed DLL3 trispecific binding proteins of the present disclosure are generated, engineered, or selected to react with selected domains, motifs, or epitopes within the DLL3 protein. In some embodiments, the DLL3 targeting trispecific protein binds to the DSL domain, and in some embodiments, binds to an epitope within the DSL domain that includes G203, R205, and P206.

[0062] The DLL3 binding domain of the DLL3-targeting trispecific proteins of the present disclosure are, in some embodiments, engineered and / or selected to react with both isoforms of DLL3 or a single isoform of the protein, or conversely, to include a pan-DLL binding domain that reacts with or associates with at least one additional DLL family member in addition to DLL3. In some embodiments, the DLL3 binding domain, e.g., the DLL3 binding domain, is engineered, engineered, and / or selected to react with a domain (or an epitope therein) that is only displayed by DLL3, or with a domain that is at least somewhat conserved across multiple or all DLL family members.

[0063] In some embodiments, the DLL3 binding domain associates with or binds to a specific epitope, portion, motif, or domain of DLL3. Both DLL3 isoforms incorporate identical extracellular regions that include at least the N-terminal domain, the DSL (Delta / Serrate / lag-2) domain, and the six EGF-like domains (i.e., EGF1-EGF6). Thus, in certain embodiments, the DLL3 binding domain associates with or binds to the N-terminal domain of DLL3 (amino acids 27-175 of the mature protein), while in other embodiments, the DLL3 binding domain associates with the DSL domain (amino acids 176-215) or an epitope therein. In other aspects of the present disclosure, the DLL3 binding domain associates with or binds to a specific epitope located in a particular EGF-like domain of DLL3. In some embodiments, the DLL3 binding domain associates with or binds to an epitope located in EGF1 (amino acids 216-249), EGF2 (amino acids 274-310), EGF3 (amino acids 312-351), EGF4 (amino acids 353-389), EGF5 (amino acids 391.427), or EGF6 (amino acids 429-465). In some embodiments, each of the aforementioned domains comprises more than one epitope and / or more than one bin. In some embodiments, the DLL3 binding domain binds, reacts, or associates with a DSL domain or an epitope therein. In other embodiments, the DLL3 binding domain binds, reacts, or associates with a specific EGF-like domain or an epitope therein. In some embodiments, the DLL3 binding domain binds, reacts, or associates with an N-terminal domain or an epitope therein.

[0064] In some embodiments, the DLL3 binding protein of the present disclosure, e.g., the DLL3 binding domain of the trispecific protein of the present disclosure, binds to the full-length DLL3 protein or a fragment thereof, e.g., an epitope-containing fragment within the full-length DLL3 protein, as described above. In some cases, the epitope-containing fragment comprises an antigenic or immunogenic fragment of the DLL3 protein and its derivatives. In some embodiments, the epitope-containing fragment, including the antigenic or immunogenic fragment, is 12 amino acids or more, 20 amino acids or more, 50 or 100 amino acids or more. In some embodiments, the DLL3 fragment comprises 95% or more of the full-length protein, 90% or more, 75% or 50% or 25% or 10% or more of the full-length protein. In some embodiments, the epitope-containing fragment of DLL3, including the antigenic or immunogenic fragment, can induce a relevant immune response in a patient. Derivatives of DLL3, in some embodiments, include variants in sequence in which one or more (e.g., 1-20, e.g., 15 amino acids, or up to 20%, e.g., 10% or 5% or 1%, depending on the number of amino acids based on the full length of the protein) deletions, insertions, or substitutions are made to the DLL3 sequence provided in SEQ ID NO: 1885 (UniProtKB Accession Q9NYJ7). In some embodiments, the substitutions include conservative substitutions. Derivatives and variants of DLL3, in some instances, have essentially the same biological function as the DLL3 protein from which they are derived. For example, derivatives and variants of DLL3, in some instances, are relatively antigenic or immunogenic to the protein from which they are derived, have either the ligand-binding activity or active receptor-complex formation ability, or preferably both, of the protein from which they are derived, and have the same tissue distribution as DLL3.

[0065] The design of the DLL3-targeting trispecific protein described herein allows the binding domain for the DLL3 protein to be flexible in that the binding domain for DLL3 can be any type of binding domain, including but not limited to, a domain derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some embodiments, the binding domain for DLL3 is a single chain variable fragment (scFv), a single domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of a single domain antibody derived from camelid. In other embodiments, the binding domain for DLL3 is a non-Ig binding domain, i.e., an antibody mimic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies. In further embodiments, the binding domain for DLL3 is a ligand or peptide that binds to or associates with DLL3. In yet further embodiments, the binding domain for DLL3 is a knottin. In yet further embodiments, the binding domain for DLL3 is a small molecule entity.

[0066] In some embodiments, the DLL3 binding domain binds to a protein comprising the sequence of SEQ ID NO: 1885 (UniProtKB Accession Q9NYJ7). In some embodiments, the DLL3 binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 1885 (UniProtKB Accession Q9NYJ7). In some embodiments, the DLL3 binding domain binds to a protein comprising the sequence of SEQ ID NO: 1892 or SEQ ID NO: 1893 (which is the mature extracellular domain of the DLL3 protein). In some embodiments, the DLL3 binding domain binds to a protein comprising amino acids 47-492 of SEQ ID NO: 1892. In some embodiments, the DLL3 binding domain recognizes an epitope within amino acids 47-4492 of SEQ ID NO: 1892.

[0067] In some embodiments, the DLL3 binding domain is an anti-DLL3 antibody or antibody variant. As used herein, the term "antibody variant" refers to variants and derivatives of the antibodies described herein. In certain embodiments, amino acid sequence variants of the anti-DLL3 antibodies described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-DLL3 antibodies described herein are contemplated to improve the binding affinity and / or other biological properties of the antibody. Exemplary methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues in the amino acid sequence of the antibody.

[0068] Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct has the desired characteristics, antigen binding. In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introduced into the antibody of interest, and the products are screened for the desired activity, retained / improved antigen binding, reduced immunogenicity, or improved T-cell-mediated cytotoxicity (TDCC). Conservative and non-conservative amino acid substitutions are contemplated for the preparation of antibody variants.

[0069] In another example of substitutions to create variant anti-DLL3 antibodies, one or more hypervariable region residues of a parent antibody are substituted. Generally, variants are selected based on improved desired properties compared to the parent antibody, such as increased affinity, decreased affinity, decreased immunogenicity, increased pH-dependent binding.

[0070] In some embodiments, the DLL3 binding domain of the DLL3 targeting trispecific protein is a single domain antibody specific for DLL3, such as a heavy chain variable domain (VH), a variable domain (VHH) of a llama-derived sdAb, a peptide, a ligand, or a small molecule entity. In some embodiments, the DLL3 binding domain of the DLL3 targeting trispecific protein described herein is any domain that binds to DLL3, including, but not limited to, a domain from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In certain embodiments, the DLL3 binding domain is a single domain antibody. In other embodiments, the DLL3 binding domain is a peptide. In further embodiments, the DLL3 binding domain is a small molecule.

[0071] In general, it should be noted that the term single domain antibody, as used herein in its broadest sense, is not limited to a particular biological source or a particular preparation method. A single domain antibody is an antibody whose complementarity determining region is part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that naturally lack light chains, single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. A single domain antibody can be any in the art or a future single domain antibody. A single domain antibody can be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, cow. For example, in some embodiments, single domain antibodies of the disclosure are obtained by: (1) isolation of a VHH domain of a naturally occurring heavy chain antibody; (2) expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) "humanization" of a naturally occurring VHH domain, or expression of a nucleic acid encoding such a humanized VHH domain; (4) "camelization" of a naturally occurring VH domain from any animal species, and in particular from a mammalian species such as human, or expression of a nucleic acid encoding such a camelized VH domain; (5) "camelization" of a "domain antibody" or "Dab", or expression of a nucleic acid encoding such a camelized VH domain; (6) use of synthetic or semi-synthetic techniques to prepare proteins, polypeptides, or other amino acid sequences; (7) preparation of a nucleic acid encoding a single domain antibody using techniques for nucleic acid synthesis known in the art, followed by expression of the nucleic acid obtained as above; and / or (8) any combination of one or more of the foregoing.

[0072] In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody against DLL3. As further described herein, such a VHH sequence can typically be generated or obtained by appropriately immunizing a species of llama with DLL3 (i.e. to generate an immune response and / or heavy chain antibodies against DLL3), by obtaining a suitable biological sample from said llama (such as a blood sample, a serum sample, or a sample of B cells) and generating a VHH sequence against DLL3 starting from said sample using any suitable technique known in the art.

[0073] In another embodiment, such naturally occurring VHH domains against DLL3 are obtained from a naive library of camelid VHH sequences, for example by screening such a library with DLL3 or at least one part, fragment, antigenic determinant or epitope thereof using at least one screening technique known in the art. Such libraries and techniques are described, for example, in WO99 / 37681, WO01 / 90190, WO03 / 025020 and WO03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, for example, VHH libraries are obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described in WO00 / 43507.

[0074] In a further embodiment, yet another technique for obtaining VHH sequences against DLL3 comprises appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e. to generate an immune response and / or heavy chain antibodies against DLL3), obtaining a suitable biological sample from said transgenic mammal (a blood sample, a serum sample, or a sample of B cells), and generating VHH sequences against DLL3 starting from said sample using any suitable technique known in the art. For example, for this purpose heavy chain antibody expressing rats or mice and methods and techniques such as those described in WO02 / 085945 and WO04 / 049794 can be used.

[0075] In some embodiments, the anti-DLL3 single domain antibodies of the DLL3-targeting trispecific protein comprise single domain antibodies having an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (and in particular in the framework sequences) with one or more amino acid residues occurring at the corresponding positions in a VH domain from a conventional four-chain antibody of human origin (e.g. as shown above). This can be done in any manner known in the art that will be clear to the skilled artisan, for example, based on the further description below. Again, such humanized anti-DLL3 single domain antibodies of the present disclosure can be obtained in any suitable manner known per se (i.e. as shown in points (1) to (8) above), and are therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as starting material. In some further embodiments, the single domain anti-DLL3 antibodies comprise single domain antibodies having an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain as described herein, but that has been "camelized", i.e. "camelized" by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VH domain from a conventional four-chain antibody with one or more amino acid residues occurring at the corresponding positions in the VHH domain of a heavy chain antibody. Such "camelized" substitutions are preferably inserted at amino acid positions that form and / or are present at the VH-VL interface and / or residues prominent in the so-called Camelidae family (see, for example, WO94 / 04678 and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence used as starting material or starting point for generating or designing the camelized single domain is a VH sequence, preferably from a mammal, more preferably a human VH sequence, such as a VH3 sequence.However, it should be noted that in certain embodiments, such camelized anti-DLL3 single domain antibodies of the present disclosure are obtained in any suitable manner known in the art (i.e., as indicated in points (1)-(8) above), and are therefore not strictly limited to polypeptides obtained using a naturally occurring VH domain-containing polypeptide as starting material. For example, as further described herein, both "humanization" and "camelization" are performed by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, and then altering one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" single domain antibody, respectively. This nucleic acid can then be expressed to provide the desired anti-DLL3 single domain antibody of the present disclosure. Alternatively, in other embodiments, the amino acid sequence of the desired humanized or camelized anti-DLL3 FLT3 single domain antibody of the present disclosure is designed based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known techniques of peptide synthesis. In some embodiments, a nucleotide sequence encoding the desired humanized or camelized anti-DLL3 single domain antibody of the disclosure is designed based on the amino acid or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known techniques of nucleic acid synthesis, and the nucleic acid thus obtained is then expressed using known expression techniques to obtain the desired anti-FLT3 single domain antibody of the disclosure.

[0076] Other suitable methods and techniques for obtaining an anti-DLL3 single domain antibody of the present disclosure and / or a nucleic acid encoding the anti-DLL3 single domain antibody starting from a naturally occurring VH or VHH sequence include, for example, combining, in a suitable manner, one or more parts of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences to provide an anti-DLL3 single domain antibody of the present disclosure or a nucleotide sequence or nucleic acid encoding the same.

[0077] In some embodiments, the DLL3 binding domain is an anti-DLL3 specific antibody comprising heavy chain variable complementarity determining region CDR1, heavy chain variable CDR2, heavy chain variable CDR3, light chain variable CDR1, light chain variable CDR2, and light chain variable CDR3. In some embodiments, the DLL3 binding domain comprises any domain that binds to DLL3, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen-binding fragment, such as a single domain antibody (sdAb), Fab, Fab', F(ab)2, and Fv fragment, a fragment composed of one or more CDRs, a single chain antibody (e.g., a single chain Fv fragment (scFv)), a disulfide-stabilized (dsFv) Fv fragment, a heteroconjugate antibody (e.g., a bispecific antibody), a pFv fragment, a heavy chain monomer or dimer, a light chain monomer or dimer, and a dimer consisting of one heavy chain and one light chain. In some embodiments, the DLL3 binding domain is a single domain antibody. In some embodiments, an anti-DLL3 single domain antibody comprises the heavy chain variable complementarity determining regions (CDRs), CDR1, CDR2, and CDR3.

[0078] In some embodiments, the DLL3 binding domain is a polypeptide comprising an amino acid sequence composed of four framework regions / sequences (f1-f4) interrupted by three complementarity determining regions / sequences, as represented by the formula: f1-r1-f2-r2-f3-r3-f4, where r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues. The framework residues of the DLL3 binding proteins of the disclosure include, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94 amino acid residues, and the complementarity determining regions include, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues. In some embodiments, the DLL3 binding domain includes an amino acid sequence selected from SEQ ID NOs: 1-442 and 1886. In some embodiments, the CDR1 of the DLL3 binding domain includes a sequence selected from SEQ ID NOs: 443-884 and 1887, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 443-884 and 1887. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 885-1326 and 1888, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 885-1326 and 1888. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 1327-1768 and 1889, or one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 1327-1768 and 1889.

[0079] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 443-884 and 1887, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 443-884 and 1887. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 885-1326 and 1888, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 885-1326 and 1888. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 1327-1768 and 1889, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in a sequence selected from SEQ ID NOs: 1327-1768 and 1889.

[0080] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 495-528, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 495-528. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 937-970, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 937-970. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 1379-1412, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in a sequence selected from SEQ ID NOs: 1379-1412.

[0081] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 529-809, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 529-809. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 971-1251, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 971-1251. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 1379-1412, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in a sequence selected from SEQ ID NOs: 1379-1412.

[0082] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 810-884, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 810-884. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 1252-1326, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in an amino acid sequence selected from SEQ ID NOs: 1252-1326. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 1692-1768, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in a sequence selected from SEQ ID NOs: 1692-1768.

[0083] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 1-442 and 1886. In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs:53-86.

[0084] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs:87-367.

[0085] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:68, or to a sequence derived from SEQ ID NO:68.

[0086] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:75, or to a sequence derived from SEQ ID NO:75.

[0087] In some embodiments, the DLL3 binding domain of the DLL3-targeting trispecific binding protein is cross-reactive with human and cynomolgus DLL3. In some embodiments, the DLL3 binding domain is specific for human DLL3. In certain embodiments, the DLL3 binding domain disclosed herein binds to human DLL3 with a human Kd (hKd). In certain embodiments, the DLL3 binding domain disclosed herein binds to cynomolgus DLL3 with a cynomolgus Kd (cKd). In certain embodiments, the DLL3 binding domain disclosed herein binds to both cynomolgus DLL3 and human DLL3 with a cynomolgus Kd (cKd) and human Kd (hKd), respectively. In some embodiments, the DLL3 binding protein binds to human and cynomolgus DLL3 with comparable binding affinity (i.e., the values ​​of hKd and cKd do not differ by more than ±10%). In some embodiments, the hKd and cKd range from about 0.001 nM to about 500 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 450 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 400 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 350 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 300 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 250 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 200 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 150 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 100 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 90 nM. In some embodiments, the hKd and cKd are in the range of about 0.2 nM to about 80 nM. In some embodiments, the hKd and cKd are in the range of about 0.3 nM to about 70 nM. In some embodiments, the hKd and cKd are in the range of about 0.4 nM to about 50 nM.In some embodiments, the hKd and cKd are in the range of about 0.5 nM to about 30 nM. In some embodiments, the hKd and cKd are in the range of about 0.6 nM to about 10 nM. In some embodiments, the hKd and cKd are in the range of about 0.7 nM to about 8 nM. In some embodiments, the hKd and cKd are in the range of about 0.8 nM to about 6 nM. In some embodiments, the hKd and cKd are in the range of about 0.9 nM to about 4 nM. In some embodiments, the hKd and cKd are in the range of about 1 nM to about 2 nM.

[0088] In certain embodiments, the DLL3 binding domain of the present disclosure preferentially binds membrane-bound DLL3 over soluble DLL3. Membrane-bound DLL3 refers to the presence of DLL3 in or on the cell membrane surface of a cell expressing DLL3. Soluble DLL3 refers to DLL3 that is no longer present in or on the cell membrane surface of a cell that expresses or has expressed DLL3. In certain examples, soluble DLL3 is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the DLL3 binding protein binds membrane-bound DLL3 at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more than soluble DLL3. In one embodiment, the antigen binding protein of the present disclosure preferentially binds membrane-bound DLL3 30-fold or more than soluble DLL3. The determination of preferential binding of an antigen binding protein to membrane-bound DLL3 over soluble DLL3 can be readily determined using assays well known in the art.

[0089] In some embodiments, any of the aforementioned DLL3 binding domains (e.g., the anti-DLL3 single domain antibodies of SEQ ID NOs: 1-442 and 1886) are tagged with an affinity peptide to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his (SEQ ID NO: 1819).

[0090] In some embodiments, any of the aforementioned DLL3 binding domains (e.g., the anti-DLL3 single domain antibodies of SEQ ID NOs: 1-442 and 1886) are tagged with an affinity peptide to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his (SEQ ID NO: 1819).

[0091] Incorporation into chimeric antigen receptors (CARs) The DLL3 targeting trispecific antigen binding protein of the present disclosure can be incorporated into a chimeric antigen receptor (CAR) in certain examples. Engineered immune effector cells, T cells or NK cells can be used to express a CAR comprising an anti-DLL3 targeting trispecific protein containing an anti-DLL3 single domain antibody as described herein. In one embodiment, a CAR comprising an anti-DLL3 targeting trispecific protein as described herein is linked to a transmembrane domain via a hinge region and to a functional signaling domain obtained from a co-stimulatory domain, OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB. In some embodiments, the CAR further comprises a sequence encoding an intracellular signaling domain such as 4-1BB and / or CD3 zeta.

[0092] Tumor growth suppression properties In certain embodiments, the disclosed DLL3-targeting trispecific proteins inhibit tumor cell proliferation in vivo when administered to a subject having tumor cells expressing DLL3. Measurement of inhibition of tumor cell proliferation can be determined by several different methods well known in the art. Non-limiting examples include direct measurement of tumor dimensions, measurement of resected tumor masses and comparison with control subjects, measurement by imaging techniques (e.g., CT or MRI) with or without the use of isotopes or luminescent molecules (e.g., luciferase) to enhance analysis, and the like.

[0093] In certain embodiments, administration of a trispecific protein of the present disclosure results in at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% inhibition of tumor cell proliferation in vivo compared to a control antigen binding agent, with about 100% inhibition of tumor proliferation indicating complete remission or disappearance of the tumor. In further embodiments, administration of a trispecific protein of the present disclosure results in about 50-100%, about 75-100%, or about 90-100% inhibition of tumor cell proliferation in vivo compared to a control antigen binding agent. In further embodiments, administration of a trispecific protein of the present disclosure results in about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100% inhibition of tumor cell proliferation in vivo compared to a control antigen binding agent.

[0094] Modification of DLL3-targeting trispecific proteins The DLL3-targeting trispecific target proteins described herein encompass derivatives or analogs in which (i) amino acids are replaced with amino acid residues not encoded by the genetic code, (ii) the mature polypeptide is fused to another compound, such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence, or a sequence for purification of the protein.

[0095] Exemplary modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, e.g., arginylation, and ubiquitination.

[0096] Modifications may occur anywhere in the DLL3-targeting trispecific proteins described herein, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. Certain common peptide modifications useful for modifying DLL3-targeting trispecific proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blocking of amino or carboxyl groups, or both, in the polypeptide by covalent modification, and ADP-ribosylation.

[0097] In some embodiments, derivatives of the DLL3-targeting trispecific proteins described herein include immunoreactive modulator derivatives and antigen binding molecules that contain one or more modifications.

[0098] In some embodiments, the trispecific DLL3 binding molecules of the present disclosure are monovalent or multivalent (bivalent, trivalent, etc.). As used herein, the term "valency" refers to the number of potential target binding sites associated with an antibody. Each target binding site specifically binds to one target molecule, or to a particular position or locus on a target molecule. If an antibody is monovalent, each binding site on the molecule specifically binds to one antigen position or epitope. If an antibody contains more than one target binding site (multivalent), each target binding site may specifically bind to the same molecule or different molecules (e.g., may bind to different ligands or different antigens, or different epitopes or positions on the same antigen).

[0099] In some embodiments, the disclosed DLL3-targeting trispecific proteins include one or more additional amino acid residue substitutions, mutations and / or modifications that result in compounds with favorable characteristics, including, but not limited to, altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased production, altered Fc ligand binding to Fc receptors (FcRs), enhanced or decreased "ADCC" (antibody-dependent cell-mediated cytotoxicity) or "CDC" (complement-dependent cytotoxicity) activity, altered glycosylation and / or disulfide bonds, and altered binding specificity. In some cases, these DLL3-targeting trispecific protein variants are advantageously used to enhance the effective anti-neoplastic properties of the disclosed DLL3-targeting trispecific proteins.

[0100] In some embodiments, the DLL3-targeting trispecific proteins of the present disclosure have a half-life in a mammal, e.g., a human or a cynomolgus monkey, of less than about 5 days, about 5 days, more than about 5 days, more than 10 days, more than about 15 days, more than about 20 days, more than about 25 days, more than about 30 days, more than about 35 days, more than about 40 days, more than about 45 days, more than about 2 months, more than about 3 months, more than about 4 months, or more than about 5 months. An increased half-life can, in some cases, result in higher serum titers, thereby reducing the frequency of administration of the DLL3-targeting trispecific protein, reducing the concentration of the antibody administered, or both.

[0101] Still other embodiments include DLL3-targeting trispecific binding proteins that contain one or more engineered glycoforms, i.e., modified glycosylation patterns or modified carbohydrate compositions covalently attached to the protein. Engineered glycoforms are useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function, increasing the affinity of the trispecific protein for a target, or facilitating production of the trispecific protein in some cases. In certain embodiments where reduced effector function is desired, the molecule is engineered to express an aglycosylated form. Substitutions that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site, are included in some embodiments. Conversely, enhanced effector function or improved binding is imparted to the Fc-containing trispecific proteins of the present disclosure in some cases by engineering in one or more additional glycosylation sites.

[0102] The DLL3 targeting proteins are optionally differentially modified during or after production by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, etc. Any of a number of chemical modifications are performed by techniques including, but not limited to, specific chemical cleavage with cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, and the like.

[0103] Various post-translational modifications encompassed by the present disclosure include, for example, N-linked or O-linked glycans, N- or C-terminal processing, attachment of chemical moieties to the amino acid backbone, chemical modification of N-linked or O-linked glycans, and addition or deletion of N-terminal methionine residues upon expression in prokaryotic host cells. Additionally, the DLL3-targeting trispecific binding proteins are optionally modified with a detectable label, such as an enzymatic, fluorescent, radioisotope, or affinity label, to allow for detection and isolation of the modulators.

[0104] Polynucleotides encoding DLL3-targeting trispecific proteins In some embodiments, polynucleotide molecules are also provided that encode the anti-DLL3 trispecific binding proteins described herein.In some embodiments, the polynucleotide molecules are provided as DNA constructs.In other embodiments, the polynucleotide molecules are provided as messenger RNA transcripts.

[0105] The polynucleotide molecule is constructed by known methods, such as by combining genes encoding the three binding domains, separated by peptide linkers or in other embodiments directly linked by peptide bonds, into one genetic construct operably linked to a suitable promoter and, optionally, a suitable transcription terminator, and expressing it in bacteria or other suitable expression systems, such as CHO cells. In embodiments in which the DLL3 binding domain is a small molecule, the polynucleotide comprises genes encoding the CD3 binding domain and the half-life prolonging domain. In embodiments in which the half-life prolonging domain is a small molecule, the polynucleotide comprises genes encoding the domains that bind to CD3 and DLL3. Depending on the vector system and host utilized, any number of suitable transcription and translation elements may be used, including constitutive and inducible promoters. The promoter is selected to facilitate expression of the polynucleotide in the respective host cell.

[0106] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment. The recombinant vector can be constructed according to known methods. Among others, vectors of interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc.), and cosmids.

[0107] A variety of expression vector / host systems can be utilized to contain and express the polynucleotides encoding the polypeptides of the described trispecific antigen binding proteins. Examples of expression vectors for expression in E. coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0108] Thus, the DLL3-targeting trispecific proteins described herein are produced, in some embodiments, by introducing vectors encoding such proteins into host cells and culturing the host cells under conditions whereby the protein domains can be expressed, isolated, and optionally further purified.

[0109] Pharmaceutical Compositions In some embodiments, pharmaceutical compositions are also provided, comprising the anti-DLL3 trispecific binding protein described herein, a vector comprising a polynucleotide encoding a polypeptide of a DLL3-targeting trispecific protein, or a host cell transformed with this vector, and at least one pharma- ceutically acceptable carrier. The term "pharma- ceutically acceptable carrier" includes, but is not limited to, a carrier that does not interfere with the effectiveness of the biological activity of the components and is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile liquids, and the like. Such carriers can be formulated by conventional methods and administered to a subject in an appropriate dosage. Preferably, the compositions are sterilized. These compositions may also contain adjuvants such as preservatives, emulsifiers, and dispersing agents. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents. Further embodiments provide one or more of the above DLL3-targeting trispecific proteins packaged in lyophilized form or packaged in an aqueous medium.

[0110] In some embodiments of the pharmaceutical composition, the DLL3 targeting trispecific protein described herein is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a fullerene, a liquid crystal, a liposome, a quantum dot, a superparamagnetic nanoparticle, a dendrimer, or a nanorod. In other embodiments of the pharmaceutical composition, the DLL3 targeting trispecific protein is bound to a liposome. In some examples, the DLL3 targeting trispecific protein is conjugated to the surface of the liposome. In some examples, the DLL3 trispecific antigen binding protein is encapsulated in the shell of the liposome. In some examples, the liposome is a cationic liposome.

[0111] The DLL3 targeting trispecific proteins described herein are intended for use as pharmaceutical agents. Administration is accomplished by a variety of methods, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of treatment and the type of compound contained in the pharmaceutical composition. The administration regimen is determined by the attending physician and other clinical factors. The dosage for a patient depends on many factors, including the patient's size, body surface area, age, sex, the specific compound administered, the time and route of administration, the type of treatment, health status, and other drugs administered concomitantly. An "effective amount" refers to an amount of active ingredient sufficient to affect the course and severity of a disease, thereby causing a reduction or remission of such pathology, and can be determined using known methods.

[0112] In some embodiments, the DLL3-targeting trispecific proteins of the present disclosure are administered at a dosage of up to 10 mg / kg once a week. In some cases, the dosage ranges from about 1 ng / kg to about 10 mg / kg. In some embodiments, the dosage ranges from about 1 ng / kg to about 10 ng / kg, about 5 ng / kg to about 15 ng / kg, about 12 ng / kg to about 20 ng / kg, about 18 ng / kg to about 30 ng / kg, about 25 ng / kg to about 50 ng / kg, about 35 ng / kg to about 60 ng / kg, about 45 ng / kg to about 70 ng / kg, about 65 ng / kg to about 85 ng / kg, about 80 ng / kg to about 1 μg / kg, about 0. 5 μg / kg to about 5 μg / kg, about 2 μg / kg to about 10 μg / kg, about 7 μg / kg to about 15 μg / kg, about 12 μg / kg to about 25 μg / kg, about 20 μg / kg to about 50 μg / kg, about 35 μg / kg to about 70 μg / kg, about 45 μg / kg to about 80 μg / kg, about 65 μg / kg to about 90 μg / kg, about 85 μg to about 0.1 mg / kg, and about 0.095 mg / kg to about 10 mg / kg. In some cases, the dosage is about 0.1 mg / kg to about 0.2 mg / kg, about 0.25 mg / kg to about 0.5 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.75 mg / kg to about 3 mg / kg, about 2.5 mg / kg to about 4 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 6 mg / kg, about 5.5 mg / kg to about 7 mg / kg, about 6.5 mg / kg to about 8 mg / kg, about 7.5 mg / kg to about 9 mg / kg, or about 8.5 mg / kg to about 10 mg / kg. The frequency of administration is, in some embodiments, about less than daily, every other day, less than once a day, twice a week, every week, once every 7 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, or about once a month. In some cases, the frequency of administration is weekly. In some cases, the frequency of administration is weekly and the dosage is up to 10 mg / kg. In some cases, the duration of administration is from about 1 day to about 4 weeks or longer.

[0113] In some embodiments, the DLL3-targeting trispecific protein of the disclosure is about 1 μg to about 100 μg, about 1 μg to about 500 μg, about 1 μg to about 1 mg, about 1 μg to about 2 mg, about 1 μg to about 5 mg, about 1 μg to about 10 mg, about 1 μg to about 100 mg, about 100 μg to about 500 μg, about 100 μg to about 1 mg, about 100 μg to about 2 mg, about 100 μg to about 5 mg, about 100 μg to about 10 mg, about 100 μg to about 100 mg , about 500 μg to about 1 mg, about 500 μg to about 2 mg, about 500 μg to about 5 mg, about 500 μg to about 10 mg, about 500 μg to about 100 mg, about 1 mg to about 2 mg, about 1 mg to about 5 mg, about 1 mg to about 10 mg, about 1 mg to about 100 mg, about 2 mg to about 5 mg, about 2 mg to about 10 mg, about 2 mg to about 100 mg, about 5 mg to about 10 mg, about 5 mg to about 100 mg, or about 10 mg to about 100 mg. In some embodiments, the DLL3-targeting trispecific protein of the present disclosure is administered at a dose of about 15 μg to about 45 μg, about 15 μg to about 135 μg, about 15 μg to about 405 μg, about 15 μg to about 1215 mg, about 15 μg to about 3600 μg, about 45 μg to about 135 μg, about 45 μg to about 405 mg, about 45 μg to about 1215 μg, about 45 μg to about 3600 μg, about 135 μg to about 405 μg, about 135 μg to about 1215 μg, about 135 μg to about 3600 μg, about 405 μg to about 1215 μg, about 405 μg to about 3600 μg, or about 1215 μg to about 3600 μg. In some embodiments, the first dose is about 5 mg. In some embodiments, the dose is about 7 mg. In some embodiments, the dose is about 10 mg. In some embodiments, the dose is about 12 mg. In some embodiments, the dose is about 15 mg. In some embodiments, the dose is about 20 mg. In some embodiments, the dose is about 30 mg. In some embodiments, the dose is about 40 mg. In some embodiments, the dose is about 50 mg. In some embodiments, the dose is about 70 mg. In some embodiments, the dose is about 100 mg.

[0114] The DLL3 targeting trispecific protein described herein can be administered using various dosages. In some embodiments, the DLL3 targeting trispecific protein of the present disclosure is administered according to a schedule that includes (i) administering a first dose of the DLL3 targeting trispecific protein, (ii) administering a second dose of the DLL3 targeting trispecific protein, where the second dose is higher than the first dose. In some embodiments, the schedule further includes (iii) administering a third dose of the DLL3 targeting trispecific protein, where the third dose is higher than the second dose. In some embodiments, the schedule further includes (iv) administering a fourth dose of the DLL3 targeting trispecific protein, where the fourth dose is higher than the third dose. In some embodiments, the schedule further includes (v) administering a fifth dose of the DLL3 targeting trispecific protein, where the fifth dose is higher than the fourth dose.

[0115] In some embodiments, the first dose is from about 1 μg to about 100 μg, from about 1 μg to about 500 μg, from about 1 μg to about 1 mg, from about 1 μg to about 2 mg, from about 1 μg to about 5 mg, from about 1 μg to about 5 mg, from about 1 μg to about 8 mg, from about 1 μg to about 10 mg, from about 1 μg to about 50 mg, from about 1 μg to about 100 mg, from about 100 μg to about 500 μg, Approximately 100μg to approximately 1mg, approximately 100μg to approximately 2mg, approximately 100μg to approximately 5mg, approximately 100μg to approximately 5mg, approximately 100μg to approximately 8mg, approximately 100μg to approximately 10 mg, about 100μg to about 50mg, about 100μg to about 100mg, about 500μg to about 1mg, about 500μg to about 2mg, about 500μg to about 5mg, about 500μ g to about 5 mg, about 500 μg to about 8 mg, about 500 μg to about 10 mg, about 500 μg to about 50 mg, about 500 μg to about 100 mg, about 1 mg to about 2 mg, about 1 mg to about 5 mg, about 1 mg to about 8 mg, about 1 mg to about 10 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, about 2 mg to about 5 mg, about 2 mg to about 8 mg, about 2 mg to about 10 mg, about 2 mg to about 50 mg, about 2 mg to about 100 mg, about 5 mg to about 8 mg, about 5 mg to about 10 mg, about 5 mg to about 50 mg, about 5 mg to about 100 mg, about 8 mg to about 10 mg, about 8 mg to about 50 mg, about 8 mg to about 100 mg, about 10 mg about 50 mg, and about 50 mg to about 100 mg. In some embodiments, the first dose is about 5 μg. In some embodiments, the first dose is about 15 μg. In some embodiments, the first dose is about 45 μg. In some embodiments, the first dose is about 135 μg. In some embodiments, the first dose is about 405 μg. In some embodiments, the first dose is about 1215 μg. In some embodiments, the first dose is about 1500 μg. In some embodiments, the first dose is about 2000 μg. In some embodiments, the first dose is about 2500 μg. In some embodiments, the first dose is about 3600 μg. In some embodiments, the first dose is about 3 mg. In some embodiments, the first dose is about 4 mg. In some embodiments, the first dose is about 5 mg. In some embodiments, the first dose is about 6 mg. In some embodiments, the first dose is about 7 mg. In some embodiments, the first dose is about 8 mg.In some embodiments, the first dose is about 9 mg. In some embodiments, the first dose is about 10 mg. In some embodiments, the first dose is about 11 mg. In some embodiments, the first dose is about 12 mg. In some embodiments, the first dose is about 15 mg. In some embodiments, the first dose is about 20 mg. In some embodiments, the first dose is about 30 mg. In some embodiments, the first dose is about 40 mg. In some embodiments, the first dose is about 50 mg. In some embodiments, the first dose is about 70 mg. In some embodiments, the first dose is about 100 mg.

[0116] In some embodiments, the first dose is from about 1 week to about 5 weeks, from about 1 week to about 10 weeks, from about 1 week to about 20 weeks, from about 1 week to about 50 weeks, from about 1 week to about 80 weeks, from about 1 week to about 100 weeks, from about 5 weeks to about 10 weeks, from about 5 weeks to about 20 weeks, from about 5 weeks to about 50 weeks, from about 5 weeks to about 80 weeks, from about 5 weeks to about 100 weeks, from about 10 weeks to about 20 weeks, from about 10 weeks to about 50 weeks, from about 10 weeks to about 80 weeks, from about 10 weeks to about 100 weeks, or from about 20 weeks. The compound is administered for about 1 to about 50 weeks, about 20 to about 80 weeks, about 20 to about 100 weeks, about 50 to about 80 weeks, about 50 to about 100 weeks, about 80 to about 100 weeks, about 1 to about 9 weeks, about 1 to about 18 weeks, about 1 to about 27 weeks, about 1 to about 36 weeks, about 9 to about 18 weeks, about 9 to about 27 weeks, about 9 to about 36 weeks, about 18 to about 27 weeks, about 18 to about 36 weeks, or about 27 to about 36 weeks.

[0117] In some embodiments, the first dose is administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, eight times a day, nine times a day, or ten times a day. In some embodiments, the first dose is administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, once every two weeks, once every three weeks, once every four weeks, or once every five weeks.

[0118] In some embodiments, the second dose is from about 1 μg to about 100 μg, from about 1 μg to about 500 μg, from about 1 μg to about 2 mg, from about 1 μg to about 2 mg, from about 1 μg to about 5 mg, from about 1 μg to about 5 mg, from about 1 μg to about 8 mg, from about 1 μg to about 10 mg, from about 1 μg to about 50 mg, from about 1 μg to about 100 mg, from about 100 μg to about 500 μg, Approximately 100μg to approximately 1mg, approximately 100μg to approximately 2mg, approximately 100μg to approximately 5mg, approximately 100μg to approximately 5mg, approximately 100μg to approximately 8mg, approximately 100μg to approximately 10 mg, about 100μg to about 50mg, about 100μg to about 100mg, about 500μg to about 1mg, about 500μg to about 2mg, about 500μg to about 5mg, about 500μ g to about 5 mg, about 500 μg to about 8 mg, about 500 μg to about 10 mg, about 500 μg to about 50 mg, about 500 μg to about 100 mg, about 1 mg to about 2 mg, about 1 mg to about 5 mg, about 1 mg to about 8 mg, about 1 mg to about 10 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, about 2 mg to about 5 mg, about 2 mg to about 8 mg, about 2 mg to about 10 mg, about 2 mg to about 50 mg, about 2 mg to about 100 mg, about 5 mg to about 8 mg, about 5 mg to about 10 mg, about 5 mg to about 50 mg, about 5 mg to about 100 mg, about 8 mg to about 10 mg, about 8 mg to about 50 mg, about 8 mg to about 100 mg, about 10 mg about 50 mg, and about 50 mg to about 100 mg. In some embodiments, the second dose is about 1.2 mg. In some embodiments, the second dose is about 2 mg. In some embodiments, the second dose is about 3 mg. In some embodiments, the second dose is about 4 mg. In some embodiments, the second dose is about 5 mg. In some embodiments, the second dose is about 6 mg. In some embodiments, the second dose is about 7 mg. In some embodiments, the second dose is about 8 mg. In some embodiments, the second dose is about 9 mg. In some embodiments, the second dose is about 10 mg. In some embodiments, the second dose is about 11 mg. In some embodiments, the second dose is about 12 mg. In some embodiments, the second dose is about 13 mg. In some embodiments, the second dose is about 14 mg. In some embodiments, the second dose is about 15 mg. In some embodiments, the second dose is about 20 mg.In some embodiments, the second dose is about 30 mg. In some embodiments, the second dose is about 40 mg. In some embodiments, the second dose is about 50 mg. In some embodiments, the second dose is about 70 mg. In some embodiments, the second dose is about 100 mg. In some embodiments, the second dose is about 3.6 mg. In some embodiments, the second dose is about 7.2 mg. In some embodiments, the second dose is about 12 mg. In some embodiments, the second dose is about 24 mg. In some embodiments, the second dose is about 36 mg. In some embodiments, the second dose is about 48 mg. In some embodiments, the second dose is about 60 mg. In some embodiments, the second dose is about 72 mg. In some embodiments, the second dose is about 84 mg. In some embodiments, the second dose is about 96 mg.

[0119] In some embodiments, the second dose is from about 1 week to about 5 weeks, from about 1 week to about 10 weeks, from about 2 weeks to about 20 weeks, from about 1 week to about 50 weeks, from about 1 week to about 80 weeks, from about 1 week to about 100 weeks, from about 5 weeks to about 10 weeks, from about 5 weeks to about 20 weeks, from about 5 weeks to about 50 weeks, from about 5 weeks to about 80 weeks, from about 5 weeks to about 100 weeks, from about 10 weeks to about 20 weeks, from about 10 weeks to about 50 weeks, from about 10 weeks to about 80 weeks, from about 10 weeks to about 100 weeks, or from about 20 weeks. The compound is administered for about 1 to about 50 weeks, about 20 to about 80 weeks, about 20 to about 100 weeks, about 50 to about 80 weeks, about 50 to about 100 weeks, about 80 to about 100 weeks, about 1 to about 9 weeks, about 1 to about 18 weeks, about 1 to about 27 weeks, about 1 to about 36 weeks, about 9 to about 18 weeks, about 9 to about 27 weeks, about 9 to about 36 weeks, about 18 to about 27 weeks, about 18 to about 36 weeks, or about 27 to about 36 weeks.

[0120] In some embodiments, the second dose is administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, eight times a day, nine times a day, or ten times a day. In some embodiments, the first dose is administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, once every two weeks, once every three weeks, once every four weeks, or once every five weeks.

[0121] In some embodiments, the first dose is about 3.6 mg, and the second dose is about 7.2 mg. In some embodiments, the first dose is about 3 mg, and the second dose is about 14 mg, and is administered every week. In some embodiments, the first dose is about 3 mg, and the second dose is about 7 mg, and is administered every week. In some embodiments, the first dose is about 3 mg, and the second dose is about 7 mg, and is administered every two weeks.

[0122] Treatment Method In some embodiments, the DLL3 binding proteins or DLL3-targeting trispecific proteins of the present disclosure are administered to treat neoplastic diseases, which in some embodiments may be benign or malignant, solid tumors or other hematological neoplasms, and in some embodiments may be any of the following: adrenal gland tumors, AIDS-related cancers, alveolar soft part sarcoma, astrocytic tumors, autonomic ganglion tumors, bladder cancer (squamous cell carcinoma and transitional cell carcinoma), blastocoel lesions, bone cancer (ameloblastoma, aneurysmal bone cyst, osteochondroma, osteosarcoma), brain and spinal cord cancer, metastatic brain tumors, breast cancer, including triple-negative breast cancer, carotid globe tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, ependymoma, epithelial disorders, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibroplasia imperfecta osseous, fibroblastoma ... ossium), fibrous dysplasia of bone, gallbladder and bile duct cancer, stomach cancer, gastrointestinal disease, gestational trophoblastic disease, germ cell tumors, glandular disorders, head and neck cancer, cancer of the hypothalamus, intestinal cancer, islet cell tumors, Kaposi's sarcoma, kidney cancer (nephroblastoma, papillary renal cell carcinoma), leukemia, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, liver cancer (hepatoblastoma, hepatocellular carcinoma), lymphoma, lung cancer (small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc.), macrophage disorders, medulloblastoma, melanoma, meningioma, multiple endocrine adenoma, multiple myeloma, The cancer is selected from the group including, but not limited to, myelodysplastic syndromes, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare blood disorders, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, stromal disorders, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer (cervical carcinoma, endometrial carcinoma, and leiomyoma).

[0123] In certain embodiments, the DLL3 binding proteins or DLL3 targeting trispecific proteins of the present disclosure are used as frontline therapy and are administered to subjects who have not been previously treated for cancer disease. In other embodiments, the DLL3 targeting trispecific proteins of the present disclosure are used to treat subjects who have been previously treated (with the DLL3 targeting trispecific proteins of the present disclosure or with other anti-cancer agents) and have relapsed or been determined to be refractory to previous treatments. In some embodiments, the DLL3 targeting trispecific proteins of the present disclosure are used to treat subjects with recurrent tumors.

[0124] In some embodiments, the DLL3 binding proteins or DLL3-targeting trispecific proteins of the present disclosure are administered to treat proliferative disorders including, but not limited to, adrenal, liver, kidney, bladder, breast, stomach, ovary, cervical, uterine, esophageal, colon, prostate, pancreas, lung (both small cell and non-small cell), thyroid, carcinoma, sarcoma, glioblastoma, and various head and neck tumors.

[0125] In some embodiments, the DLL3 binding protein or DLL3 targeting trispecific protein of the present disclosure is administered to a subject suffering from melanoma. In some embodiments, the DLL3 targeting trispecific protein of the present disclosure is used to diagnose, monitor, treat, or prevent melanoma. The term "melanoma" as used herein includes all types of melanoma, including but not limited to primary melanoma, malignant melanoma, cutaneous melanoma, extracutaneous melanoma, superficial spreading melanoma, polypoid melanoma, malignant melanoma, melanoepithelioma, melanosarcoma, in situ melanoma, nodular malignant melanoma, lentigo maligna melanoma, lentigo melanoma, mucosal lentigo melanoma, mucosal melanoma, acral lentigo melanoma, soft tissue melanoma, ocular melanoma, invasive melanoma, familial atypical lentigo and melanoma (FAM-M) syndrome, desmoplastic malignant melanoma, or uveal melanoma.

[0126] DLL3 is an effective tumor marker that has been found to be expressed in many different cancers and associated with cancer stem cells. Thus, in some embodiments where the disclosed DLL3-binding proteins or DLL3-targeting trispecific proteins are integrated into chimeric antigen receptors expressed on lymphocytes, the resulting "DLL3-sensitized lymphocytes" (e.g., natural killer cells or T cells that immunospecifically recognize the DLL3 determinant) can effectively mount an immune response directed against abnormal DLL3-positive cells, including cancer stem cells. This ability to effectively eliminate tumorigenic "seed" cells is often crucial in reducing the likelihood of tumor recurrence or metastasis. In some embodiments, such DLL3-sensitized lymphocytes are used in combination with other therapeutic agents or as part of a conservative regimen following standard treatment.

[0127] More generally, a chimeric antigen receptor is an artificially constructed hybrid protein or polypeptide that encompasses or includes an antigen-binding domain of an antibody linked to a signaling domain (e.g., a T cell signaling domain or a T cell activation domain). In some embodiments, the DLL3-targeting trispecific binding protein of the present disclosure has the ability to redirect the specificity and reactivity of sensitized lymphocytes (e.g., T cells) toward DLL3-positive target cells in a non-MHC-restricted manner by utilizing the antigen-binding properties of an antibody or its antigen-binding fragment. Non-MHC-restricted antigen recognition can provide T cells expressing DLL3CAR with the ability to recognize tumorigenic DLL3 independent of antigen processing, thus circumventing a major mechanism of tumor escape. Moreover, when expressed in T cells, the CAR advantageously does not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.

[0128] In selected embodiments, the DLL3 binding proteins, or DLL3-targeting trispecific proteins of the present disclosure are incorporated into a chimeric antigen receptor (CAR) and the DLL3 CAR is administered in a CAR-based therapy effective to treat lung cancer, including the following subtypes: small cell lung cancer, non-small cell lung cancer (e.g., squamous cell non-small cell lung cancer or squamous cell small cell lung cancer), and large cell neuroendocrine carcinoma (LCNEC).

[0129] In some embodiments, the DLL3 binding protein or DLL3-sensitive lymphocytes are administered to patients exhibiting limited or extensive disease stages. In some embodiments, the disclosed DLL3-targeting trispecific antibodies are administered to refractory patients (i.e., patients whose disease recurs during or shortly after the completion of the initial course of therapy), to sensitive patients (i.e., patients whose recurrence is longer than 2-3 months after the first-line treatment), or to patients exhibiting resistance to platinum-based drugs (e.g., carboplatin, cisplatin, oxaliplatin) and / or taxanes (e.g., docetaxel, paclitaxel, larotaxel, or cabazitaxel). In another embodiment, the disclosed DLL3 CAR treatment is effective in treating ovarian cancer, including ovarian serous carcinoma and ovarian papillary serous carcinoma.

[0130] The disclosed DLL3 binding proteins, or DLL3-targeting trispecific binding proteins, are used in some embodiments to prevent, treat, or diagnose tumors with neuroendocrine characteristics or phenotypes, including neuroendocrine tumors. True or canonical neuroendocrine tumors (NETs), arising from the dispersed endocrine system, are relatively rare with an incidence of 2-5 per 100,000, but are highly aggressive. Neuroendocrine tumors arise in the kidney, genitourinary tract (bladder, prostate, ovaries, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lung (small cell lung carcinoma and large cell neuroendocrine carcinoma). These tumors may secrete several hormones, including serotonin and / or chromogranin A, which may cause the debilitating condition known as carcinoid syndrome. Such tumors can be indicated by positive immunohistochemical markers such as neuron-specific enolase (NSE, also known as gamma enolase, gene symbol=ENO2), CD56 (or NCAM1), chromogranin A (CHGA), and synaptophysin (SYP), or by genes known to show elevated expression such as ASCL1. Conventional chemotherapy has not been particularly effective in treating neuroendocrine tumors, and liver metastasis is a common outcome. In some embodiments, the DLL3-targeting trispecific antibodies of the present disclosure are advantageously used to treat neuroendocrine tumors, and in some embodiments, they are used to treat, prevent, or diagnose pseudoneuroendocrine tumors (pNETs), which genotypically or phenotypically mimic, resemble, or display traits in common with canonical neuroendocrine tumors. Pseudoneuroendocrine tumors, or tumors with neuroendocrine features, arise from disseminated neuroendocrine cells or from cells that have aberrantly reactivated the neuroendocrine differentiation cascade during the tumorigenic process.Such pNETs share certain phenotypic or biochemical characteristics with classically defined neuroendocrine tumors, including the ability to generally produce a subset of biologically active amines, neurotransmitters, and peptide hormones.Histologically, such tumors (NETs and pNETs) share a common appearance, often showing small, densely contiguous cells with minimal cytoplasm and round to oval, speckled nuclei that are cytopathologically bland. In some embodiments of the present disclosure, commonly expressed histological or genetic markers used to determine neuroendocrine tumors and pseudoneuroendocrine tumors include, but are not limited to, chromogranin A, CD56, synaptophysin, PGP9.5, ASCL1, and neuron-specific enolase (NSE). Thus, in some embodiments, the disclosed DLL3-targeting trispecific proteins, DLL3 CARs, or DLL3-sensitized lymphocytes, or any combination thereof, are beneficially used to treat both pseudoneuroendocrine tumors and canonical neuroendocrine tumors, such as to treat neuroendocrine tumors (both NETs and pNETs) occurring in the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lung (small cell lung carcinoma and large cell neuroendocrine carcinoma). Furthermore, in some embodiments, the disclosed DLL3-targeting trispecific proteins, DLL3 CARs, or DLL3-sensitized lymphocytes, or any combination thereof, are used to treat tumors expressing one or more markers, such as NSE, CD56, synaptophysin, chromogranin A, ASCL1, or PGP9.5 (UCHL1). In some embodiments, the DLL3-targeting trispecific proteins, DLL3 CARs, or DLL3-sensitized lymphocytes of the present disclosure, or any combination thereof, are used to treat a subject suffering from a tumor that is NSE+ or CD56+ or PGP9.5+ or ASCL1+ or SYP+ or CHGA+, or any combination thereof.

[0131] In another embodiment, the disclosed DLL3-targeting trispecific protein, DLL3 CAR, or DLL3-sensitized lymphocyte, or any combination thereof, is used in maintenance therapy to reduce or eliminate the possibility of tumor recurrence following the initial symptoms of the disease. In some cases, the patient is asymptomatic or in remission because the disorder has been treated and the initial tumor mass has been removed, reduced, or otherwise improved. At such times, the subject is administered one or more pharmacologic effective amounts of the disclosed DLL3-binding protein, DLL3 CAR, or DLL3-sensitized lymphocyte, or any combination thereof, regardless of whether there are few or no signs of disease using standard diagnostic procedures. In some embodiments, the disclosed DLL3-targeting trispecific protein, DLL3 CAR, or DLL3-sensitized lymphocyte, or any combination thereof, is administered on a regular schedule for a period of time, such as every week, every two weeks, every month, every six weeks, every two months, every three months, every six months, or every year, to reduce the possibility of disease recurrence. Moreover, such treatment, in some embodiments, is continued for weeks, months, years, or even indefinitely, depending on the patient's response and clinical and diagnostic parameters.

[0132] In yet another embodiment, the disclosed DLL3 binding proteins, DLL3 targeting trispecific proteins, DLL3 CARs, or DLL3 sensitized lymphocytes, or any combination thereof, are used prophylactically or as adjuvant therapy to prevent or reduce the likelihood of tumor metastasis following a debulking treatment. As used in this disclosure, "debulking treatment" is broadly defined to mean any treatment, technique, or method that removes, reduces, treats, or improves tumor or tumor growth. Exemplary debulking treatments include, but are not limited to, surgery, radiation therapy (i.e., beam radiation), chemotherapy, immunotherapy, or ablation. In some embodiments, the disclosed DLL3 binding proteins, DLL3 targeting trispecific proteins, DLL3 CARs, or DLL3 sensitized lymphocytes, or any combination thereof, are administered at the appropriate time as suggested by clinical, diagnostic, or theranostic procedures to reduce tumor metastasis. In some embodiments, the administration regimen is accompanied by appropriate diagnostic or monitoring techniques that allow it to be modified.

[0133] Yet another embodiment of the present disclosure includes administering the DLL3 binding protein, DLL targeting trispecific protein, DLL3 CAR, or DLL3 sensitized lymphocyte, or any combination thereof, of the present disclosure to a subject who is asymptomatic but at risk of developing a proliferative disorder. That is, in some embodiments, the DLL3 binding protein, DLL targeting trispecific protein, DLL3 CAR, or DLL3 sensitized lymphocyte, or any combination thereof, of the present disclosure, is used in a prophylactic sense and is given to a patient who has been examined or tested and has one or more significant risk factors (e.g., genomic indications, family history, in vivo or in vitro test results, etc.) but has not developed neoplasia. In such cases, a person skilled in the art will be able to determine an effective dosing regimen through empirical observation or generally accepted clinical practice.

[0134] As used herein, in some embodiments, "treatment" or "treating" or "treated" refers to a therapeutic treatment aimed at delaying (alleviating) an undesirable physiological disease, disorder, or condition, or obtaining a beneficial or desired clinical outcome. For purposes described herein, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of the disease, disorder, or disease; stabilization (i.e., not worsening) of the disease, disorder, or disease state; delaying the onset or slowing the progression of the disease, disorder, or disease; amelioration of the disease, disorder, or disease state; and remission (whether partial or total) or improvement or amelioration of the disease, disorder, or disease, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment further includes extending survival time compared to expected survival time in the absence of treatment. In other embodiments, "treatment" or "treating" or "treated" refers to a preventative measure, the purpose of which is to delay the onset of or reduce the severity of an undesirable physiological disease, disorder, or condition, e.g., in an individual predisposed to the condition (e.g., an individual carrying genetic markers for a disease such as breast cancer).

[0135] In some embodiments of the methods described herein, the DLL3 binding protein, DLL3 targeting trispecific protein, or composition as described herein is administered in combination with an agent for the treatment of a particular disease, disorder, or condition. The agent includes, but is not limited to, therapeutic agents including antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiotherapy agents (directed delivery of gamma rays, X-rays, and / or radioisotopes, microwave, UV radiation, etc.), gene therapy agents (e.g., antisense, retroviral therapy, etc.), and other immunotherapeutics. In some embodiments, the anti-DLL3 binding protein or anti-DLL3 targeting trispecific protein described herein is administered in combination with an antidiarrheal, antiemetic, analgesic, opioid, and / or nonsteroidal anti-inflammatory agent. In some embodiments, the anti-DLL3 binding protein or anti-DLL3 targeting trispecific protein as described herein is administered in combination with an anti-cancer agent. Non-limiting examples of anti-cancer drugs that can be used in various embodiments of the present disclosure, including the pharmaceutical compositions and dosage forms and kits of the present disclosure, include acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, aztomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, biceresin, Bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calsterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingal, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexoromaplatin, dezaguamine, dezaguamine mesylate, diazicon, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate,Dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, fluroocitabine, foskidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride Mucitabine, hydroxyurea, idarubicin hydrochloride, ifosfamide, irmofosine, interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1 interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, and masomito Procol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocalcine, mitochromine, mitodilline, mitomarcine, mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxislan, paclitaxel, pegaspargase, periomycin, pentamustine, peplomycin sulfate, peplomycin sulfate, Rufosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, ribopurin, rogletimide, safingol, safingol hydrochloride, semustine, simtrazene, sparphosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenal, tallysomycin,These include tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfin, teniposide, teroxylone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifene citrate, trestrone acetate, tricibirine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubrozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglisinate sulfate, vinleurodine sulfate, vinorelbine tartrate, vinzoquidine sulfate, vinzoquidine sulfate, vorozole, zeniplatin, zinostatin, and zorubicin hydrochloride. Other examples of anti-cancer drugs include, but are not limited to, 20-epi-1,25 dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol, adozelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-1, protein-1), antiandrogen, prostate cancer, antiestrogens, antineoplastons, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asaculin, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorine, benzoylstaurosporine, beta-lactam derivatives, beta-arretin, betaclamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, bisaziridinylspermine, biansafide, bisstraten A, biceresin, brefullate, bropirimine, budotitanium, buthionine sulfoximine, calcipotriol,Calphostin C, camptothecin derivatives, canaripox IL-2, capecitabine, carboxamido-amino-triazole, carboxyamidotriazole, CaRest M3, CARN 700, cartilage derived inhibitor, carzelesin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorin, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene analog, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analog, conagenin, crambescidin 816, crisnatol, cryptophycin 8, crypto Ficin A derivatives, curacin A, cyclopentane tyramine, cycloplatin, sipemycin, cytarabine ocfosfate, cytotoxic factors, cytostatin, daclizumab, decitabine, dehydrodidemin B, deslorelin, dexamethasone, dexphosphamide, dexrazoxane, dexverapamil, diazicon, dididemin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, 9-, dioxamycin, diphenylspiromustine, docetaxel, docosahexaenoic acid nol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epirubicin, epristeride, estramustine analogs, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, fretinol, Gelastin, fluasterone, fludarabine, fluorodaunorhysine hydrochloride, forphenimex, formestane, fostriecin, fotemustine, gadolinium texapyrin, gallium nitrate, gallocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfame, heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, irmofosine, irmostat,Imidazoacridone, imiquimod, immunostimulant peptides, insulin-like growth factor I receptor inhibitors, interferon agonists, interferons, interleukins, iobenguane, iododoxorubicin, ipomeanol, 4-, ilopract, irsogladine, isobengazole, isohomohalichondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibitory factor, leukocyte alpha interferon lon, leuprolide + estrogen + progesterone, leuprorelin, levamisole, liarozole, linear polyamine analogs, lipophilic disaccharide peptides, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone, HMG-CoA reductase inhibitors (such as, but not limited to, lovastatin, pravastatin, fluvastatin, statins, simvastatin, and atorvastatin), loxoribine, lurtotecan, lutetium texaphyrin, lisofilis , cytolytic peptides, maytansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, menogaril, mervalone, metalelin, methioninase, metoclopramide, MIF inhibitors, mifepristone, miltefosine, millimostim, incompatible double-stranded RNA, mitoguazone, mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mofalotene, molgramostim, monoclonal antibodies (human placental gonadotropic hormones), monophosphoryl lipid A + myobacterial cell wall sk, mopidamol, multidrug resistance gene inhibitors, multiple tumor suppressor gene 1-based therapy, mustard anticancer drugs, mycaloxide B, mycobacterial cell wall extract, myriapolone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagressip, naloxone + pentazocine, napavine, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidants,Nitrulline, O6-benzylguamine, octreotide, oxenone, oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, paclitaxel, Paclitaxel analogues, paclitaxel derivatives, palaumin, palmitoyl rhizoxin, pamidronic acid, panaxytriol, panomyphen, parabactin, pazelliptin, pegaspargase, perdecin, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide, peryl alcohol, phenazinomycin, phenyl acetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, prasetin A, prasetin B, plasminogen activator inhibitors, platinum complexes, platinum compounds, platinum-triamine complexes, porfimer sodium, porf thromycin, prednisone, propyl bis-acridone, prostaglandin J2, proteasome inhibitors, protein A-based immunomodulators, protein kinase C inhibitors, microalgae-derived protein kinase C inhibitors, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurins, pyrazoloacridines, pyridoxylated hemoglobin polyoxyethylene conjugates, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitors, demethylated reteliptin, rhenium Re 186 Etidronate, Rhizoxin, Ribozyme, RII retinamide, Rogletimide, Rohitukine, Romurtide, Roquinimex, Rubiginone B1, Ruboxil, Safingol, Saintpin, SarCNU, Sarcophytol A, Sargramostim, Sdi1 mimetic, Semustine, Senessense-derived inhibitor 1, Sense oligonucleotide, Signal transduction inhibitor, Signal transduction modulator, Single-stranded antigen-binding protein, Sizofiran, Sobuzoxane, Borocaptate sodium, sodium phenylacetate, solverol, somatomedin binding protein, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongiostatin 1, squalamine, stem cell inhibitor, stem cell division inhibitor, stipiamide, stromelysin inhibitor, sulfinodine, superactive vasoactive intestinal peptide antagonist, slajista, suramin, swainsonine, synthetic glycosaminoglycan, talimustine,Tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, telluropyrylium, telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, thalicarpine, thiocoraline, thrombopoietin, thrombopoietin mimetics, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tin ethyl etioproprine, tirapazamine, titanocene dichloride, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triazoline These include cetyluridine, tricibirine, trimetrexate, triptorelin, tropisetron, tulosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitors, urokinase receptor antagonists, vapreotide, variolin B, vector systems, red blood cell gene therapy, veraresol, veramine, verdins, verteporfin, vinorelbine, vinxartin, Vitaxin®, vorozole, zanoteron, zeniplatin, zilascorub, and zinostatin stimalamer. Additional anticancer agents are 5-fluorouracil and leucovorin. These two agents are particularly useful when used in methods employing thalidomide and topoisomerase inhibitors. In some embodiments, the DLL3-targeting trispecific protein of the present disclosure is used in combination with gemcitabine. In some embodiments, the DLL3-targeting trispecific proteins described herein are administered before, during, or after surgery.

[0136] Methods for detecting DLL3 expression and diagnosing DLL3-associated cancers - Patents.com According to another embodiment of the present disclosure, a kit is provided for detecting the expression of DLL3 in vitro or in vivo. The kit includes the above-mentioned DLL3 binding protein, a DLL3 targeting trispecific protein (e.g., a trispecific protein including a labeled anti-DLL3 single domain antibody or an antigen-binding fragment thereof), and one or more compounds for detecting the label. In some embodiments, the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

[0137] In some cases, DLL3 expression is detected in biological samples.Samples can be any samples, including but not limited to tissue from biopsy, autopsy, and pathological specimens.Biological samples also include tissue sections, such as frozen sections taken for histological purposes.Biological samples also include bodily fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine.Biological samples are usually obtained from mammals, such as humans and non-human primates.

[0138] In one embodiment, a method is provided for determining whether a subject has cancer by contacting a sample from the subject with an anti-DLL3 single domain antibody or anti-DLL3 trispecific protein disclosed herein and detecting binding of the single domain antibody to the sample. Increased binding of the antibody to the sample compared to binding of the antibody to a control sample identifies the subject as having cancer.

[0139] In another embodiment, a method is provided for confirming the diagnosis of cancer in a subject by contacting a sample from a subject diagnosed with cancer with an anti-DLL3 single domain antibody or anti-DLL3 trispecific protein disclosed herein and detecting binding of the antibody to the sample. Increased binding of the antibody to the sample compared to binding of the antibody to a control sample confirms the diagnosis of cancer in the subject.

[0140] In some examples of the disclosed method, the DLL3 binding protein or the DLL3 binding single domain antibody of the trispecific protein is directly labeled. In some examples, the method further comprises contacting the sample with a second antibody that specifically binds to the anti-DLL3 single domain antibody or the anti-DLL3 trispecific protein, and detecting the binding of the second antibody. An increase in the binding of the second antibody to the sample compared to the binding of the second antibody to the control sample detects cancer in the subject or confirms the diagnosis of cancer in the subject. In some cases, the cancer is a neuroendocrine cancer, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple negative breast cancer, or ovarian cancer epithelial ovarian cancer, or any other type of cancer that expresses DLL3. In some examples, the control sample is a sample from a subject that does not have cancer. In certain examples, the sample is a blood or tissue sample.

[0141] In some cases, the antibody that binds (e.g., specifically binds) DLL3 is directly labeled with a detectable label. In another embodiment, the antibody that binds (e.g., specifically binds) DLL3 (first antibody) is unlabeled, and a second antibody or other molecule that can bind to the antibody that specifically binds DLL3 is labeled. The second antibody is selected to specifically bind to the first antibody of a particular species and class. For example, if the first antibody is a llama IgG, the second antibody may be an anti-llama IgG. Other molecules that can bind to antibodies include, but are not limited to, protein A and protein G, both of which are commercially available. Suitable labels for antibodies or second antibodies are described above and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic groups include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary fluorescent material is luminol, a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125I, 131I, 35S, or 3H.

[0142] In an alternative embodiment, DLL3 can be assayed in a biological sample by a competitive immunoassay that utilizes a DLL3 standard labeled with a detectable substance and an unlabeled antibody that specifically binds to DLL3. In this assay, the biological sample, the labeled DLL3 standard, and the antibody that specifically binds to DLL3 are combined to determine the amount of labeled DLL3 standard bound to the unlabeled antibody. The amount of DLL3 in the biological sample is inversely proportional to the amount of labeled DLL3 standard bound to the antibody that specifically binds to DLL3.

[0143] The immunoassays and methods disclosed herein can be used for many purposes. In one embodiment, the antibody that specifically binds to DLL3 can be used to detect the production of DLL3 in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of DLL3 in a biological sample, such as a tissue sample or a blood or serum sample. In some examples, the DLL3 is cell surface DLL3. In other examples, the DLL3 is soluble DLL3 (e.g., DLL3 in cell culture supernatant, or soluble DLL3 in a body fluid sample, such as a blood or serum sample).

[0144] In one embodiment, a kit is provided for detecting DLL3 in a biological sample, such as a blood sample or a tissue sample. For example, a biopsy can be performed to obtain a tissue sample for histological examination to confirm a cancer diagnosis in a subject. Alternatively, a blood sample can be obtained to detect the presence of soluble DLL3 protein or fragments. A kit for detecting a polypeptide typically includes a single domain antibody of the present disclosure that specifically binds to DLL3. In some embodiments, an antibody fragment, such as an scFv fragment, a VH domain, or a Fab, is included in the kit. In further embodiments, the antibody is labeled (e.g., with a fluorescent label, a radioactive label, or an enzyme label).

[0145] In one embodiment, the kit includes instructional materials disclosing the means of using the antibody that binds to DLL3. The instructional materials may be written, electronic (such as computer diskette or compact disk), visual (such as video files), or provided via an electronic network, such as the Internet, World Wide Web, intranet, or other network. The kit may further include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally include a means for detecting the label (e.g., an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a secondary antibody, etc.). The kit may additionally include buffers and other reagents routinely used in the practice of a particular method. Such kits and appropriate contents are well known to those skilled in the art.

[0146] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassay may vary depending on the particular format employed, the method of detecting DLL3 in a biological sample generally comprises contacting the biological sample with an antibody that specifically reacts with a DLL3 polypeptide under immunologically reactive conditions. The antibody specifically binds to form an immune complex under immunologically reactive conditions, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0147] Methods for determining the presence or absence of cell surface markers are well known in the art. For example, antibodies can be conjugated to other compounds, including but not limited to enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorescent dyes, metal compounds, radioactive compounds, or drugs. Antibodies can further be utilized in immunoassays, such as but not limited to radioimmunoassays (RIA), ELISA, or immunohistochemistry assays. Antibodies can also be used in fluorescence-activated cell sorting (FACS). FACS employs multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels, among other advanced levels of detection, to separate or sort cells (see U.S. Pat. No. 5,061,620). Any of the single domain antibodies that bind to DLL3 as disclosed herein can be used in these assays. Thus, antibodies can be used in conventional immunoassays, including but not limited to ELISA, RIA, FACS, tissue immunohistochemistry, Western blot, or immunoprecipitation. EXAMPLES

[0148] Example 1: Screening of a phage display library to identify DLL3 binding domains Llamas were immunized with purified DLL3 protein expressed in EXPI293™ cells. A phage display library for the expression of heavy chain variable antibody domains from circulating B cells was constructed (see van der Linden, de Geus, Stok, Bos, van Wassenaar, Verrips, and Frenken. 2000. J Immunol Methods 240:185-195). Phage clones were screened for binding to DLL3 by expressing the clones in E. coli, preparing periplasmic extracts, and screening the clones for DLL3 binding activity by ELISA. Fifty-two unique heavy chain only single domain antibodies were identified that generated a signal in the ELISA screen (SEQ ID NOs: 1-52). The CDR1, CDR2, and CDR3 sequences for these heavy chain variable domains were SEQ ID NOs: 443-494, SEQ ID NOs: 885-936, and SEQ ID NOs: 1327-1378, respectively.

[0149] Example 2: Humanization of DLL3-binding single domain antibodies and T-cell dependent cytotoxicity assay Thirty-four exemplary llama anti-DLL3 heavy chain only single domain antibodies (SEQ ID NOs:53-86) from Example 1 were humanized. The CDR1, CDR2, and CDR3 sequences for the 34 heavy chain only single domain antibodies were SEQ ID NOs:495-528, SEQ ID NOs:937-970, and SEQ ID NOs:1379-1412, respectively.

[0150] The humanized anti-DLL3 sequence was cloned into an expression vector and the expression construct included the signal domain followed by the anti-DLL3 heavy chain only variable domain followed by the GGGGSGGGS linker (SEQ ID NO:1808), followed by the anti-human albumin single domain antibody 10G (SEQ ID NO:1774), followed by the GGGGSGGGS linker (SEQ ID NO:1808), followed by the anti-human CD3 antibody 2B2 (SEQ ID NO:1793), followed by the HHHHHH tag (SEQ ID NO:1819) to generate the anti-DLL3 trispecific construct.

[0151] Anti-DLL3 trispecific constructs containing humanized anti-DLL3 binding sequences were then transfected into EXPI293™ cells. These anti-DLL3 trispecific constructs were engineered with a Protein A binding site, and the amount of anti-DLL3 trispecific construct in conditioned medium from transfected EXPI293™ cells was quantified using an Octet instrument equipped with a Protein A chip. Trispecific proteins of similar molecular weight to the anti-DLL3 trispecific proteins were used as standards.

[0152] The binding affinity of the anti-DLL3 trispecific protein to human and cynomolgus DLL3 protein was measured using conditioned medium containing known concentrations of the anti-DLL3 trispecific protein, where the DLL3 protein was expressed as a human IgG1-Fc fusion, and measurements were performed using an Octet instrument equipped with an anti-human Fc chip. D Measurements were made using a single 50 nM concentration of the anti-DLL3 trispecific protein, which allowed for a rank ordering based on potency. The relative affinities, measured as described above, are listed in Table 1. All sequences had relative affinities (K D ) were found to bind to human DLL3. Several of the sequences were found to bind to cynomolgus DLL3 with similar affinity to human DLL3, and the relative affinity of binding of those sequences to cynomolgus DLL3 is also shown in Table 1.

[0153] Conditioned media was also tested in a T cell dependent cytotoxicity assay (see Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27), in which luciferase-labeled DMS-153 cells (small cell lung carcinoma cell line; ATCC No. ATCC® CRL-2064™) were combined with purified human T cells from a donor and titrations of anti-DLL3 trispecific proteins were tested.

[0154] It was hypothesized that if the anti-DLL3 trispecific protein instructed T cells to kill DMS-153 cells expressing DLL3, the viability of DMS-153 cells would be decreased as determined by performing a luciferase assay at 48 hours after the initiation of the experiment.

[0155] As illustrated in Figures 2-6, which show graphs of representative TDCC data, several exemplary anti-DLL3 trispecific proteins were able to reduce the viability of DMS-153 cells. Figure 2 shows the results of a TDCC assay for an anti-DLL3 trispecific protein comprising DLL3 binding domains DH18 (SEQ ID NO: 59), DH11 (SEQ ID NO: 55), DH67 (SEQ ID NO: 42), and DH56 (SEQ ID NO: 73). Figure 3 shows the results of a TDCC assay for an anti-DLL3 trispecific protein comprising DLL3 binding domains DH2 (SEQ ID NO: 60), DH43 (SEQ ID NO: 68), DH10 (SEQ ID NO: 54), and DH6 (SEQ ID NO: 75). Figure 4 shows the results of a TDCC assay for an anti-DLL3 trispecific protein comprising DLL3 binding domains DH82 (SEQ ID NO: 81), DH23 (SEQ ID NO: 62), DH89 (SEQ ID NO: 84), and DH17 (SEQ ID NO: 58). Figure 5 shows the results of a TDCC assay for a DLL3 trispecific protein containing DLL3 binding domains DH83 (SEQ ID NO: 82), DH12 (SEQ ID NO: 56), DH61 (SEQ ID NO: 76), and DH29 (SEQ ID NO: 64). Figure 6 shows the results of a TDCC assay for a DLL3 trispecific protein containing DLL3 binding domains DH58 (SEQ ID NO: 74) and DH70 (SEQ ID NO: 79). The negative control for the TDCC assay was a trispecific protein that targets GFP instead of DLL3 (as shown in Figure 6) and does not instruct T cells to kill DMS-153 cells. ECs from the TDCC assay 50 The values ​​are also listed in Table 1. These values ​​ranged from 69 pM to 11 nM.

[0156] [Table 1]

[0157] Example 3: Screening of a phage display library to identify DLL3 binding domains with higher binding affinity using the two humanized DLL3 single domain antibodies from the above examples Two humanized antibody sequences, DH43 (SEQ ID NO: 68) and DH6 (SEQ ID NO: 75), were used as starting points to generate a phage display library (following the methods described in WO2016187101A2). The anti-DLL3 sequences from this panning were then cloned into an expression vector, and the expression construct included a signal domain, followed by an anti-DLL3 heavy chain only variable domain, followed by a GGGGSGGGS linker (SEQ ID NO: 1808), followed by an anti-human albumin single domain antibody domain, followed by a GGGGSGGGS linker (SEQ ID NO: 1808), followed by an anti-human CD3 antibody fragment, followed by a HHHHHH tag (SEQ ID NO: 1819) to generate an anti-DLL3 trispecific protein. These constructs were transfected into EXP1293™ cells, and the expressed anti-DLL3 trispecific protein was quantified as described in Example 2. The sequences of the clones identified from the panning are SEQ ID NOs: 87-367. Table 2 presents the CDR mutations obtained in the DH43 DLL3 binder sequence after phage display selection. Three clones identified from panning, SEQ ID NO: 199 (2E05), 330 (4D09), and 365 (4H011), were engineered to generate variants, where each variant had a single amino acid change from the parent sequence, for example, to remove potential metabolic trends of the parent sequence. In particular, the DLL3 binding domains including SEQ ID NO: 227 (2E05-M106Y), 228 (2E05-M106Q) were engineered variants of SEQ ID NO: 199 (2E05), SEQ ID NO: 366 (4D09-M34L) were engineered variants of SEQ ID NO: 330 (4D09), and SEQ ID NO: 367 (4H11-M34L) were engineered variants of SEQ ID NO: 365 (4H011). The CDR1 sequences of these DLL3-binding clones identified by panning are SEQ ID NOs: 529-809, the CDR2 sequences of the clones identified by panning are SEQ ID NOs: 971-1251, and the CDR3 sequences of the clones identified by panning are SEQ ID NOs: 1413-1691.

[0158] [Table 2]

[0159] Conditioned medium with known concentrations of anti-DLL3 trispecific protein was used to measure the binding affinity of anti-DLL3 trispecific protein to human DLL3 protein by expressing a biotinylated version of human DLL3 protein as a human IgG1 fusion protein and performing binding affinity measurements on an Octet instrument equipped with a streptavidin chip. D Measurements were made using a single 50 nM concentration of the anti-DLL3 trispecific protein, which allowed for a rank ordering of potency. In this experiment, the relative K D Values ​​ranged from 2.3 nM to 64 nM, as listed in Table 3. The parental binders DH43 and DH6 had K values ​​of 7.7 ± 0.6 nM and 9.9 ± 0.3 nM, respectively, based on four samples of conditioned medium from four transfections. D had value.

[0160] For selected DLL3 binder molecules identified in this round of panning, as well as the parental DLL3 binders, DH43 and DH6, more precise affinity measurements for human DLL3 were performed using 60 nM, 20 nM, 6.67 nM and 2.22 nM concentrations of the anti-DLL3 trispecific protein. In addition, relative affinity measurements were performed using only 60 nM of the anti-DLL3 trispecific protein. The binding affinities of some anti-DLL3 binding molecules determined from more precise measurements are listed in Table 4 [1H012 (SEQ ID NO: 162), 1A011 (SEQ ID NO: 95), 2E05 (SEQ ID NO: 199), 4H011 (SEQ ID NO: 365), 3C04 (SEQ ID NO: 251), 2E02 (SEQ ID NO: 198), 2H02 (SEQ ID NO: 221), 3A011 (SEQ ID NO: 238), 3A02 (SEQ ID NO: 230), 4D09 (SEQ ID NO: 330), DH43 (SEQ ID NO: 68), and DH6 (SEQ ID NO: 75)]. In this study, the parent binder, DH43, had a K of 8.9 nM. DWhile the highest affinity daughter molecule, 1H012 (SEQ ID NO: 162), had an affinity of 2.9 nM. Additionally, 1H012 (SEQ ID NO: 162) retained the ability to bind to cynomolgus DLL3. Also in this study, the parent binder, DH6, had a K of 9.0 nM. D values, whereas the highest affinity daughter molecule, 4H011 (SEQ ID NO: 365), had an affinity of 3.9 nM. Additionally, 4H011 (SEQ ID NO: 365) retained the ability to bind to cynomolgus DLL3.

[0161] Twenty-two DLL3 binder molecules identified in this round of panning were selected for testing in a TDCC assay with DMS-153 cells using the same protocol as described in Example 2. Exemplary TDCC data are plotted graphically in Figures 7-11 and show EC 50 A summary of the values ​​is listed in Table 5. In this experiment, the parental DLL3 molecules, DH43 and DH6, had EC 50 The most potent daughter molecule of DH43 was 1H012 (SEQ ID NO: 162), which had an EC 50 The EC value was 28 nM, demonstrating a more than 7-fold increase in TDCC potency compared to the parent DLL3 binder DH43. The most potent daughter molecule of DH6 was 4H011 (SEQ ID NO: 365), with an EC 50 The value was 36 nM, indicating a greater than 8-fold increase in TDC potency compared to the parent DLL3 binding molecule. A control trispecific protein targeting GFP, used as a control, had no activity in this assay (shown in FIG. 11).

[0162] [Table 3-1]

[0163] [Table 3-2]

[0164] [Table 4]

[0165] [Table 5]

[0166] Example 4: Cloning of selected DLL3 binding molecules from Example 3 into mammalian cells The anti-DLL3 trispecific proteins described in Example 3, as well as the parental DLL3 binder molecules, were subcloned into a CHO cell expression vector and stably transfected into CHO cells. (See Running Deer and Allison 2004. Biotechnol. Prog. 20:880-889). The DLL3 binder molecules were 2E05-M106Q (SEQ ID NO:228), 2C04 (SEQ ID NO:181), 4D09-M34L (SEQ ID NO:366), 4D09 (SEQ ID NO:330), 2E05-M106Y (SEQ ID NO:227), 1H012 (SEQ ID No.162) (also referred to herein as 1H12), 2E05 (SEQ ID NO:199), 2H02 (SEQ ID NO:221), 4D011 (SEQ ID NO:332) (also referred to herein as 4D11), 2E02 (SEQ ID NO:198), 4H11-M34L (SEQ ID NO:367), 1A011 (SEQ ID NO:95) (also referred to herein as 1A11), DH6 (SEQ ID NO:75), and DH43 (SEQ ID NO:68). The anti-DLL3 trispecific protein was expressed in CHO cells in conditioned medium from a pool of stable clones and then purified using protein A and ion exchange chromatography. The purified protein was tested in a TDCC assay using the same method as described in Example 2. ECs from the TDCC assay in this example 50 The values ​​are listed in Table 6 and graphs of the data are shown in Figures 12-15. The most potent molecule, 2E05-M106Q (SEQ ID NO: 228), had an EC of 41 nM, 6.6-fold more potent than the parent molecule, DH43. 50 The most potent molecule derived from DH6 was 4D09-M34L (SEQ ID NO: 366), which had an EC 50value, being 4.4-fold more potent than the parent molecule, DH6.

[0167] [Table 6]

[0168] Example 5: Affinity maturation to obtain anti-DLL3 binders with improved affinity To obtain stronger anti-DLL3 binders, a second round of affinity maturation was performed. A phage display library was made based on the parent sequences of DH6 (SEQ ID NO: 75) and DH58 (SEQ ID NO: 74). The sequences of the binders from this round of affinity maturation are presented in SEQ ID NOs: 368-442. The CDR1 sequences of the DLL3 binders identified in this round of affinity maturation are SEQ ID NOs: 810-884, the CDR2 sequences of the DLL3 binders identified in this round of affinity maturation are SEQ ID NOs: 1252-1326, and the CDR3 sequences of the DLL3 binders identified in this round of affinity maturation are SEQ ID NOs: 1692-1768. Table 7 presents the CDR variants obtained in the DH46 DLL3 binder sequence after phage display selection.

[0169] The affinity matured anti-DLL3 sequences identified above were cloned into an expression vector such that the expression construct included the signal domain, followed by the anti-DLL3 sequence, followed by the GGGGSGGGS linker (SEQ ID NO:1808), followed by the anti-human albumin single domain antibody 10G (SEQ ID NO:1774), followed by the GGGGSGGGS linker (SEQ ID NO:1808), followed by the anti-human CD3 antibody 2B2 (SEQ ID NO:1793), followed by the HHHHHH tag (SEQ ID NO:1819) to generate the anti-DLL3 trispecific construct.

[0170] Anti-DLL3 trispecific constructs containing affinity matured anti-DLL3 binding sequences were then transfected into EXPI293™ cells. These anti-DLL3 trispecific constructs were then engineered with protein A binding sites, and the amount of anti-DLL3 trispecific constructs in conditioned medium from transfected EXP1293™ cells was quantified using an Octet instrument equipped with a protein A chip. A control trispecific protein of similar molecular weight to the anti-DLL3 trispecific protein was used as a standard.

[0171] Conditioned media with known concentrations of anti-DLL3 trispecific protein was used to measure the relative binding affinity of anti-DLL3 trispecific protein to human DLL3 protein by expressing a biotinylated version of human DLL3 protein as a human IgG1 fusion protein and performing binding affinity measurements on an Octet instrument equipped with a streptavidin chip. D Measurements were performed using a single 50 nM concentration of the anti-DLL3 trispecific protein, allowing a ranking of potencies. The affinities measured are shown in Table 8. All sequences tested had K values ​​ranging from 0.3 nM to 34 nM. D It was found to bind to human DLL3 at 100 ng / mL.

[0172] Conditioned media was also tested in a T cell-dependent cytotoxicity assay (see Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27). In this assay, luciferase-labeled DMS-153 cells were combined with purified human T cells and a titration of anti-DLL3 trispecific protein. It was hypothesized that if the anti-DLL3 trispecific protein instructed T cells to kill DLL3-expressing DMS-153 cells, the viability of DMS-153 cells would be decreased as determined by performing a luciferase assay 48 hours after the start of the experiment. 16 illustrates a graph depicting TDCC data for anti-DLL3 trispecific proteins that include the following DLL3 binding domains: 51A02 (SEQ ID NO: 409), 51G02 (SEQ ID NO: 425), 52B01 (SEQ ID NO: 430), 52C04 (SEQ ID No. 431), 51A05 (SEQ ID NO: 411), 52D04 (SEQ ID NO: 432), 51E05 (SEQ ID NO: 420), 51H05 (SEQ ID NO: 429), purified DH43 protein (SEQ ID NO: 68), and purified DH6 protein (SEQ ID NO: 75). EC from TDCC assay 50 The values ​​are listed in Table 9. Values ​​ranged from 4.2 pM to 1.5 nM. The negative control for the TDCC assay was a fused trispecific protein that targets GFP (shown in FIG. 16), but does not instruct T cells to kill DMS-153 cells.

[0173] [Table 7]

[0174] [Table 8-1]

[0175] [Table 8-2]

[0176] [Table 8-3]

[0177] [Table 9-1]

[0178] [Table 9-2]

[0179] [Table 9-3]

[0180] Example 6: Affinity maturation to obtain anti-DLL3 binders with improved affinity The specific anti-DLL3 trispecific proteins including the DLL-3 binding sequences with the most potent TDCC activity in the assay described in Example 5, and the anti-DLL3 trispecific proteins including the parent DLL3 binder DH6 were subcloned into CHO cell expression vectors and stably transfected into CHO cells (see Running Deer and Allison 2004. Biotechnol. Prog. 20:880-889). The DLL3 binding sequences were DH6 (SEQ ID NO:75), 51A2 (SEQ ID NO:408), 51A5 (SEQ ID NO:411), 51F3 (SEQ ID NO:423), 51G2 (SEQ ID NO:425), 51G10 (SEQ ID NO:427), 51H5 (SEQ ID NO:429), 51X5 (SEQ ID NO:1886), 52B1 (SEQ ID NO:430), 52C4 (SEQ ID NO:431), and 52D4 (SEQ ID NO:432). The trispecific protein was purified from the stable clone pool into conditioned media using Protein A and ion exchange chromatography. An SDS-PAGE image of the purified protein is provided in FIG.

[0181] Affinity measurements for human and cynomolgus DLL3 were made using 60 nM, 20 nM, 6.67 nM, and 2.22 nM concentrations of biotinylated DLL3 targeting the trispecific protein immobilized on an Octet streptavidin chip. The affinities determined from the measurements are listed in Table 10. In this experiment, the anti-DLL3 trispecific, which encompasses DH6, the parental DLL3 binder sequence to the affinity matured DLL3 binder sequence, had a K of 13.5 nM for human DLL3 and 11 nM for cynomolgus DLL3. D In comparison, the 10 anti-DLL3 trispecific proteins, including the affinity matured DLL3 binding molecules tested in this experiment, had K values ​​ranging from 0.9 to 2.2 nM for human DLL3 and 1.4 to 3.4 nM for cynomolgus DLL3. D Thus, the affinity improvements ranged from 6.1-15 fold for human DLL3 and 3.2-7.9 fold for cynomolgus DLL3.

[0182] The purified proteins were tested in TDCC assays using the same methods as described in Example 2, except that two additional DLL3-expressing cell lines, DMS-53 and NCI-H510A, were included in the assay. The ECs from these TDCC assays 50 The values ​​are listed in Table 11 and graphs of the DMS-53 and DMS-153 TDCC data are shown in Figures 18-19, respectively. Trispecific molecules targeting GFP had no activity in these assays (as shown in Figures 18-19). Compared to the parent molecule DH6, the EC 50 Values ​​improved by 2.3-12.1-fold in DMS-153 cells, 4.5-31.5-fold in NCI-H510A cells, and 8.1-26.1-fold in DMS-153 cells.

[0183] [Table 10]

[0184] [Table 11]

[0185] Example 7: T cell dependent cell cytotoxicity assay using exemplary DLL3-targeting trispecific proteins including DLL3 binding proteins of the present disclosure Several exemplary DLL3 trispecific proteins encompassing the disclosed DLL3 binding domain 52D04 (SEQ ID NO: 432) were tested in a T-cell dependent cytotoxicity (TDCC) assay (see Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27) and the results are shown in Figures 22-24. The trispecific proteins encompassed the DLL3 binding domain, the albumin binding domain (anti-ALB), and the CD3 binding domain (anti-CD3) in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration as shown in Figure 20, or in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration as shown in Figure 21. The TDCC assay was performed in the presence or absence of 15 mg / ml human serum albumin (HSA). In this assay, luciferase-labeled NCI-H2171 cells (FIG. 22), DMS-79 cells (FIG. 23), SHP77 cells (FIG. 24), or WM2664 cells (FIG. 25) in the presence or absence of albumin were combined with purified human T cells and titrations of the exemplary DLL3-binding trispecific proteins. It was hypothesized that if the DLL3 trispecific proteins instructed T cells to kill DLL3-expressing NCI-H2171, DMS-79, SHP77, or WM2664 cells, the viability of those cells would be decreased as determined by performing a luciferase assay 48 hours after the start of the experiment. FIG. 22 illustrates a graph of representative TDCC data for DLL3-binding trispecific proteins in the TAC or CAT configurations encompassing the following DLL3-binding domains using NCI-H2171 cells: Figure 23 illustrates a graph of representative TDCC data for DLL3-binding trispecific proteins in the TAC or CAT configurations containing the following DLL3-binding domains using DMS-79 cells: Figure 24 illustrates a graph of representative TDCC data for DLL3-binding trispecific proteins in the TAC or CAT configurations encompassing the following DLL3-binding domains using SHP77 cells:25 illustrates a representative TDCC data graph for DLL3-binding trispecific proteins in the TAC or CAT configurations encompassing the following DLL3-binding domains using WM2664 cells: EC from TDCC assay. 50 The values ​​are listed in Table 12 and Table 1. As shown in the graph, and EC 50 As shown by the values, in the presence of human serum albumin (HSA), the DLL3-binding trispecific protein with the CAT orientation (FIG. 21) was more potent than the DLL3-binding trispecific protein with the TAC configuration in the TDCC assay.

[0186] [Table 12]

[0187] Example 8: Binding of an exemplary DLL3-targeting trispecific protein to human T cells In cell binding studies, human T cells were incubated in the presence or absence of an exemplary DLL3-targeting trispecific protein (either anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration (SEQ ID NO: 1891) or anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration (SEQ ID NO: 1890)). Human T cells were further incubated with a secondary antibody (anti-trispecific antibody) capable of recognizing the anti-albumin domain in the exemplary trispecific molecule and conjugated to Alexa Fluor 647. Anti-trispecific antibody binding was measured by flow cytometry. Robust binding of the anti-trispecific antibody was seen in the presence of the exemplary DLL3 trispecific protein in the anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration (right peak in the plot in FIG. 26) compared to cells incubated with the secondary antibody alone or with the exemplary trispecific protein or without the secondary antibody (left peak in the plot in FIG. 26). Robust binding of the anti-trispecific antibody was also observed in the presence of the exemplary DLL3 trispecific protein in the anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration (right peak in the plot in FIG. 27) compared to cells incubated with the secondary antibody alone or with the exemplary trispecific protein or no secondary antibody (left peak in the plot in FIG. 27).

[0188] Example 9: Binding of exemplary DLL3-targeting trispecific proteins to DLL3-expressing cancer cell lines In another binding study, DLL3-expressing cancer cells [NCI-H82 (lung cancer cell line), SHP77 (lung cancer cell line), DMS53 (lung cancer), or NCI-H2171 (lung cancer cell line)] were incubated with an exemplary DLL3-targeting trispecific molecule (in the CAT or TAC configuration; SEQ ID NO: 1890 and SEQ ID NO: 1891) or a control GFP-targeting trispecific molecule. Following incubation, the cells were washed to remove unbound trispecific molecules and further incubated with a secondary antibody capable of recognizing the anti-albumin domain in the trispecific molecule and conjugated to Alexa Fluor 647 or FITC. Binding of the exemplary DLL3-targeting trispecific molecule or the control trispecific molecule to the cells was measured by flow cytometry. Robust binding of the DLL3-targeted trispecific (in the TAC configuration) to each cell was observed (right peak in the plot in FIG. 28) compared to cells cultured with a control GFP-targeted trispecific molecule (left peak in the plot in FIG. 28). Robust binding of the DLL3-targeted trispecific (in the CAT configuration) to each cell was observed (right peak in the plot in FIG. 29) compared to cells cultured with a control GFP-targeted trispecific molecule (left peak in the plot in FIG. 29). In control experiments with cell lines lacking DLL3 expression, HCTI16 (a colon cancer cell line), NCI-H292 (a lung cancer cell line), similar amounts of anti-trispecific antibodies bound to cells cultured with the exemplary DLL3-targeted trispecific protein or GFP-targeted suppression (data not shown), and the exemplary DLL3-targeted trispecific molecule did not bind to cells lacking DLL3 expression.

[0189] Example 10: Ability of exemplary DLL3-targeting trispecific proteins to induce T cell-mediated killing of DLL3-expressing cancer cell lines The goal of this study was to evaluate whether the exemplary DLL3-targeting trispecific molecules were able to induce T cells to kill the DLL3-expressing cell lines NCI-H82, SHP77, DMS53, and NCI-H2171. The DLL3-expressing cells used in this study were engineered to express luciferase.

[0190] For TDCC assays (T-cell dependent cytotoxicity assays), T cells and DLL3-expressing cells from four healthy donors (donor 2, donor 47, donor 81, donor 86) were mixed and various amounts of exemplary DLL3-targeting trispecific proteins (in the CAT or TAC configuration; SEQ ID NO:1890 and SEQ ID NO:1891) were added to the mixture. The mixtures were incubated at 37° C. for 48 hours. As a control, a parallel experiment was performed using a control trispecific molecule targeting GFP. After 48 hours, the amount of viable DLL3-expressing cells remaining was quantified using a luminescence assay. The DLL3-targeting trispecific molecule (in both the TAC and CAT configurations) was able to effectively induce T cells from all four healthy donors to kill all four DLL3-expressing cell lines (see Figures 30, 31, 32, and 33 for results using the TAC configuration; see Figures 34, 35, 36, and 37 for results using the CAT configuration), whereas the control GFP TriTAC molecule was not able to (also shown in Figures 30-37). 50 The values ​​are shown in Tables 13 and 14. Further TDCC assays were performed using DLL3-targeted TriTAC and the cell lines NCI-H292 and HCT116 lacking DLL3 expression. It was observed that DLL3-targeted TriTAC was unable to induce T cells to kill these two cell lines lacking DLL3 expression (data not shown).

[0191] [Table 13]

[0192] [Table 14]

[0193] Example 11: DLL3-dependent T cell activation by exemplary DLL3-targeting trispecific proteins In this assay, T cells from four different healthy donors (donor 2, donor 35, donor 47, and donor 86) and NCI-H82 or DMS53 cells were incubated with an exemplary DLL3-targeting trispecific protein (in either the CAT or TAC configuration; SEQ ID NO:1890 and SEQ ID NO:1891) for 48 hours at 37 °C. T cells from the same donors were also incubated with a control trispecific molecule, GFP TriTAC targeting GFP, and NCI-H82 or DMS53 cells for 48 hours at 37 °C. After 48 hours, T cells were collected and expression of CD69 and CD25 in T cells was measured by flow cytometry. An increase in CD69 or CD25 expression was detected in T cells from all four healthy donors in the presence of NCI-H82 or SHP77 cells and the DLL3-targeting trispecific molecule, but not in the presence of the negative control GFP TriTAC, as can be seen in Figures 38-45. Parallel experiments were also performed with HCT116 cells, which lack expression of DLL3. No increase in CD69 or CD25 expression was observed with the DLL3 trispecific molecule tested with HCT116 cells (data not shown).

[0194] Example 12: DLL3-dependent T cell cytokine production by exemplary DLL3-targeting trispecific proteins In this assay, NCI-H82 or SHP77 cells from healthy donors were incubated with the exemplary 3-targeting trispecific molecule (in either the CAT or TAC configuration; SEQ ID NO:1890 and SEQ ID NO:1891) for 48 hours at 37°C. T cells from the same donors were also incubated with a control trispecific molecule, GFP TriTAC targeting GFP, along with NCI-H82 or DMS53 cells for 48 hours at 37°C. After 48 hours, conditioned medium was collected and the amount of various cytokines in the conditioned medium was measured using an electrochemiluminscent assay (Meso Scale Discovery). Secretion of IFNγ, IL-2, and TNFα into the medium in the presence of NCI-H82 or SHP77 cells and the DLL3-targeting trispecific molecule, but not in the presence of the control GFP-targeting TriTAC molecule, was observed. For DLL3-targeted trispecific molecules in the TAC configuration, IFNγ production is shown in Figures 46 and 47, IL-2 production is shown in Figures 48 and 49, and TNFα production is shown in Figures 50 and 51. For DLL3-targeted trispecific molecules in the CAT configuration, IFNγ production is shown in Figures 52 and 53, IL-2 production is shown in Figures 54 and 55, and TNFα production is shown in Figures 56 and 57.

[0195] Example 13: Inhibition of NCI-H82 xenograft growth by exemplary DLL3-targeted trispecific proteins For this study, 5 × 10 6 of human T cells and 5 × 10 6of NCI-H82 small cell lung cancer cells were injected into mice on day 0. From days 1 to 10, mice were injected intraperitoneally (ip) daily with exemplary DLL3-targeting trispecific molecules (in either CAT or TAC configuration; SEQ ID NO: 1890 and SEQ ID NO: 1891) at doses of 20, 100, or 500 μg / kg or with negative control GFP-targeting TriTAC at a dose of 500 μg / kg. Tumor volumes were measured every 2-3 days starting on day 7 and ending on day 24. Significant inhibition of tumor growth was observed at all doses in mice injected with DLL3-targeting trispecific proteins compared to mice receiving GFP-targeting TriTAC dosed at 500 μg / kg, as shown in FIG. 58.

[0196] Example 14: Elimination of NCI-H82 xenografts with exemplary DLL3-targeted trispecific proteins For this study, 5 × 10 6 NCI-H82 small cell lung cancer cells were injected subcutaneously on day 0. Mice were randomized on day 8, with 2 × 10 cells per mouse. 7 of human T cells. From day 9 to day 18, mice were injected intraperitoneally daily with an exemplary DLL3-targeting trispecific molecule (in the CAT configuration; SEQ ID NO: 1890) at doses of 1, 10, or 100 μg / kg, or with a negative control GFP-targeting TriTAC at a dose of 100 μg / kg. Tumor volumes were measured every 2-3 days starting on day 8 and ending on day 29. Significant inhibition of tumor growth was observed in mice injected with the DLL3-targeting trispecific protein at doses of 10 and 100 μg / kg compared to mice receiving GFP-targeting TriTAC dosed at 100 μg / kg, as shown in FIG. 59.

[0197] Example 15: Inhibition of SHP77 xenograft growth by exemplary DLL3-targeted trispecific proteins For this study, 5 × 10 6 1 × 10 human T cells and 7SHP77 small cell lung cancer cells were injected into mice on day 0. From days 1 to 10, mice were injected ip daily with DLL3-targeting trispecific molecule (in the CAT construct; SEQ ID NO: 1890) at doses of 1, 10, or 100 μg / kg or with negative control GFP-targeting TriTAC at a dose of 100 μg / kg. Tumor volumes were measured every 2-3 days starting on day 6 and ending on day 28. Significant inhibition of tumor growth was observed in mice injected with DLL3-targeting trispecific protein at doses of 10 and 100 μg / kg compared to mice receiving GFP-targeting TriTAC at 100 μg / kg, as shown in FIG. 60.

[0198] Example 16: Pharmacokinetic Profiles of Exemplary DLL3-Targeted Trispecific Proteins The DLL3-targeted trispecific protein has a half-life of about 3 to about 3.9 days in cynomolgus monkeys when dosed at 0.3 mg / kg.

[0199] For this study, cynomolgus monkeys were intravenously injected with an exemplary DLL3-targeting trispecific molecule (in CAT or TAC configuration; SEQ ID NO: 1890 and SEQ ID NO: 1891) at a dose of 0.3 mg / kg, and serum samples were collected at various time points after injection. Two monkeys were injected with each dose. The amount of DLL3-targeting trispecific molecule in serum was measured using an anti-idiotypic antibody that recognizes the trispecific molecule in an electrochemiluminescient assay. Figure 61 shows a plot of serum DLL3-targeting trispecific molecule levels at various time points. The data was then used to calculate the pharmacokinetic properties of the DLL3-targeting trispecific molecule, as presented in Table 15. A once-twice-weekly human dosing schedule was contemplated based on the pharmacokinetic data.

[0200] [Table 15]

[0201] The DLL3 trispecific protein has a half-life of about 2.8 to about 3.3 days in cynomolgus monkeys when dosed at 1 or 10 mg / kg.

[0202] For this study, cynomolgus monkeys were injected intravenously with an exemplary DLL3-targeted trispecific molecule at a dose of 1 mg / kg or 10 mg / kg, and serum samples were collected at various time points after injection. Two monkeys were injected with each dose. The amount of DLL3-targeted TriTAC in serum was measured using an anti-idiotypic antibody that recognizes the TriTAC molecule in an electrochemiluminescient assay. Figure 62 shows a plot of serum DLL3-targeted trispecific molecule levels at various time points. The data was then used to calculate the pharmacokinetic properties of the TriTAC molecule, as presented in Table 16. The pharmacokinetic data suggests once or twice weekly dosing in humans.

[0203] [Table 16]

[0204] Exemplary DLL3-targeted trispecific proteins were tolerated in cynomolgus monkeys when given as single doses of up to 10 mg / kg:

[0205] Transient increases in serum cytokine levels were observed primarily at the 10 mg / kg dosage of administration of the exemplary DLL3-targeting trispecific protein (in the CAT configuration) (Figure 63; IFNγ-Figure 63 top panel, IL-6 Figure 63 second panel; IL-10 Figure 63 third panel). Transient T cell margination and T cell activation were also observed (not shown). No macroscopic findings or organ weight differences associated with the DLL3 trispecific protein were observed at terminal and convalescent euthanasia, and no microscopic findings associated with the DLL3 trispecific protein were observed at convalescent euthanasia.

[0206] To demonstrate that DLL3-targeted TriTAC retains cell-directed killing activity after administration to cynomolgus monkeys, serum samples from the 10mg / kg dose group taken 168 hours after administration were tested in the DMS53 TDCC assay and compared to freshly thawed DLL3-targeted TriTAC. DMS53 cell killing was observed similarly for serum samples and freshly thawed protein (Figure 64), indicating that DLL3-targeted TriTAC retains the ability to induce T cells to kill target cells one week after administration to cynomolgus monkeys.

[0207] Example 17: Xenograft tumor model Exemplary anti-DLL3 targeted trispecific proteins of the present disclosure are evaluated in xenograft models.

[0208] Female immunodeficient NOD / scid mice were sublethally irradiated (2 Gy) and given 1 × 10 6 NCI-H28 cells were inoculated subcutaneously into the right dorsal flank. 3 Once the population reaches 100,000, animals are assigned to one of three treatment groups: Groups 2 and 3 (8 animals each) receive 1.5×10 7 of activated human T cells are injected intraperitoneally. Three days later, animals in group 3 are subsequently administered an exemplary DLL3 trispecific antigen binding protein (such as 1, 10, 50, or 100 μg / kg) intravenously for a total of nine times (qdx9d). Groups 1 and 2 are treated with vehicle only. Body weight and tumor volume are measured for 30 days.

[0209] It is expected that animals treated with the DLL3-targeted trispecific antigen binding proteins in the previous examples will have a statistically significant delay in tumor growth compared to the respective vehicle-treated control groups.

[0210] Example 18: Proof-of-concept clinical trial protocol for administration of an exemplary DLL3 trispecific antigen binding protein (anti-DLL3 trispecific protein) to patients with neuroendocrine cancer This is a Phase I / II clinical trial investigating DLL3 trispecific antigen binding protein as a treatment for neuroendocrine cancer.

[0211] Research results:

[0212] First: Maximum Tolerated Dose of Exemplary DLL3-Targeted Trispecific Proteins

[0213] Second: To determine whether in vitro responses of exemplary DLL3-targeting trispecific proteins correlate with clinical responses

[0214] Phase I

[0215] The maximum tolerated dose (MTD) will be determined in the Phase I section of the study. 1.1 The maximum tolerated dose (MTD) will be determined in the Phase I section of the study. 1.2 Patients who meet the eligibility criteria will be enrolled in a study evaluating an exemplary DLL3-targeted trispecific protein. 1.3 The goal is to identify the maximum dose of an exemplary DLL3-targeted trispecific protein that can be safely administered without severe or unmanageable side effects in participants. The dose given will depend on the number of participants enrolled in the previous study and how well they tolerate the dose. Not all participants will receive the same dose.

[0216] Phase II 2.1 In the Phase II section that follows, treatment will be performed at the MTD with the goal of determining whether treatment with an exemplary DLL3-targeted trispecific protein results in a response rate of at least 20%. Primary outcomes for Phase II---determine whether at least 20% of patients achieve a clinical response (blow-up response, minor response, partial response, or complete response) as a result of DLL3-targeted trispecific protein treatment.

[0217] Eligibility: Biopsy proven neuroendocrine tumor with positive somatostatin receptor PET.

[0218] All sites or origins are eligible.

[0219] This applies to both functioning and non-functioning tumors.

[0220] Not a candidate for surgical debulking.

[0221] ECOG performance status 0, 1 or 2

[0222] Age > 18

[0223] Be able to understand and willing to sign a written informed consent.

[0224] Example 19: DLL3 trispecific antigen binding protein phase 1 / 2a dose escalation, expansion, safety and pharmacokinetic study Target population: Patients with small cell lung cancer (SCLC) who have relapsed after platinum-based chemotherapy or other malignancies with high-grade neuroendocrine features who are relapsed / refractory (R / R) to standard of care (SOC) or for whom SOC is unavailable, including neuroendocrine prostate cancer (NEPC) and other neuroendocrine neoplasms (NEN).

[0225] Study objectives: To assess safety and tolerability at increasing doses, determine PK and pharmacodynamic data, and evaluate preliminary antitumor activity.

[0226] Study Design: The DLL3 trispecific antigen binding protein Phase 1 / 2a study design is shown in Figure 65. The objectives of the study are to evaluate safety and tolerability with escalating doses, determine pK and pharmacodynamic data, and evaluate preliminary antitumor activity.

[0227] Dosage and Administration: DLL3 trispecific antigen binding protein (SEQ ID NO: 1890) was administered at EC 50The treatment was started at 15 μg (flat dose), equivalent to 100 mg / kg / day, administered intravenously once weekly. A cycle is 21 days including 3 doses. Patients were premedicated with dexamethasone, Tylenol, and a histamine receptor antagonist at the time of the first dose. Table 17 shows the treatment cohorts and number of subjects. The weekly dosing was well tolerated, with no dose-limiting toxicities (DLTs) observed to date. Table 18 shows the baseline demographics of these patients. The median number of prior systemic therapies was 2, with a range of 1 to 5. 77.8% of patients had prior exposure to immune checkpoint inhibitors, including 100% of patients with SCLC.

[0228] [Table 17]

[0229] [Table 18]

[0230] Treatment time: The median treatment duration was 11.6 weeks, with a range of 4.1 to 41.4 weeks. Six in 10 patients (33%) were on treatment for 20 weeks or more. Figure 66 shows the duration of treatment, weekly dose, and number of previous treatments for patients.

[0231] Safety and tolerability: No dose-limiting toxicities (DLTs) were observed. Grade 1-2 CRS was reported in 4 (22%) patients, and no grade ≥3 CRS was reported. No immune effector cell-associated neurotoxicity syndrome (ICANS) was reported. No patients discontinued due to adverse events.

[0232] [Table 19]

[0233] Target Lesion Response: Seven of 18 patients (38.9%) had a reduction in the sum of target lesion diameters (five with SCLC, one with NEPC, and one with NEN (atypical thymic carcinoid)). One patient with SCLC (2L) had a confirmed partial response and is still on treatment at 32 weeks. In patients with SCLC, three of 11 patients (27.3%) had a reduction in the sum of target lesion diameters of 30% or more across all doses. Six of 18 patients (33%) had a best overall response with stable disease, including one with SCLC, one with NEPC, and one with NEN.

[0234] Figure 67 shows the maximum percent target lesion response from baseline in each cohort.

[0235] Patient 102 Profile: Patient 102 is a 71 year old female diagnosed with SCLC in September 2020. Treatment was initiated at 45 ng / kg and demonstrated a 38% reduction in unconfirmed partial response (PR) at week 9 (Figure 68). Patient 102 has not been observed to have any treatment-related adverse events (AEs) to date and remains on study treatment beyond week 9.

[0236] [Table 20]

[0237] Figure 69 illustrates the pharmacokinetic data of DLL3 trispecific antigen binding protein for different dosing cohorts. With dose escalation, an increased half-life of approximately 70 hours and an increased serum Cmax were observed.

[0238] Figure 70 demonstrates the results of flow analysis. Figure 70A demonstrates T cell margination levels after treatment, which shows that there is dose-dependent and transient peripheral T cell margination. Figure 70B demonstrates activation marker induction after treatment. T cell activation was observed in the 135 μg / week cohort, supporting in vivo T cell activation.

[0239] Patient 111 Profile: Patient 111 is a 61-year-old female diagnosed with extensive SCLC in January 2021. Selected target lesion (TL) metastases are 1 in the lung, 2 in the liver, and 2 in lymph nodes. Non-target lesion (non-TL) metastases are 2 in the lung and 2 in the liver. Upfront systemic treatment included carboplatin, etoposide, and atezolizumab for 20.1 weeks. At study entry, stable disease was the best response to most recent prior therapy. Treatment was initiated at 1215 μg / week, escalated to 3600 μg / week, C3D15 initiated (week 8), and then escalated to 7000 μg / week. A partial response (PR) was confirmed at week 10, with a 53.3% reduction in the sum of target lesion diameters, and the patient has been on treatment for >32 weeks.

[0240] [Table 21]

[0241] Figure 71A demonstrates the change in target lesions over time for patient 111. The CT scan in Figure 71B illustrates the reduction in the sum of target lesion diameters for patient 111. Target lesion diameters were reduced by 38.1% at 6 weeks post-treatment and 53.3% at 10 weeks post-treatment.

[0242] Patient 112 Profile: Patient 112 is a 67-year-old male diagnosed with extensive SCLC in April 2020. TL metastases are 2 in the liver and 2 in lymph nodes. Non-TL are in the liver, lymph nodes, spleen, bone, and brain. Prior systemic treatment included 4 cycles of carboplatin, etoposide, toripalimab (anti-PD1), 2 cycles of cisplatin, etoposide, and lurbinectin in clinical trials. The most recent systemic treatment history was 10.9 weeks. At study entry, a partial response was the best response to the most recent prior systemic treatment. Patient 112 was treated with step-dose (3,600 μg / week, then 7,200 μg / week). At week 9, there was a 27% reduction in the sum of the diameters of target lesions, which were primarily in lymph nodes, liver metastases were stable, symptoms improved, and the patient is continuing treatment beyond week 10. At week 27, the sum of the target lesion diameters was reduced by 64.6% from baseline, with patient 112 continuing treatment beyond week 28.

[0243] [Table 22]

[0244] Figure 72A demonstrates the change in target lesions over time for patient 112. The CT scan in Figure 72B illustrates the decrease in the sum of the target lesion diameters for patient 112.

[0245] Patient 113 Profile: Patient 113 is a 65-year-old male diagnosed with neuroendocrine prostate cancer in November 2020. TL metastases are 2 in the lung, 1 in the liver, and 2 in lymph nodes. Non-TL metastases are in the lung, liver, lymph nodes, and prostate. Prior treatments include cisplatin and etoposide, and CAV. History of most recent systemic treatment is 4 weeks. At study initiation, progressive disease was the best response to most recent prior treatment. Patient 113 received stepped doses of treatment (3600 μg / week, then 7200 μg / week). At week 9, there was a 15.3% reduction in the sum of the diameters of the target lesions, shrinkage of the lung lesions and prostate was observed, new lesions were identified in the liver, urinary symptoms and pain were significantly reduced, quality of life improved, and the patient continues on study beyond week 10. Figure 73 shows the change in target lesions over time for patient 113.

[0246] Pharmacokinetics. The DLL3 trispecific antigen binding protein used in this study has linear PK with a dose-proportional increase in exposure from 0.135 to 12 mg, and a median half-life of 71 hours.

[0247] Figure 74AB shows the concentration-time profile (Figure 74A) and Cmax by dose (Figure 74B).

[0248] Pharmacokinetics. T cell margination was observed, consistent with target engagement. Small, transient increases in serum IL-6 and MCP-1 were observed up to 24 hours post-dose. A "first dose" effect was observed with less margination, and lower median IL-6 and MCP-1 concentrations were observed with repeat or targeted doses.

[0249] FIG. 75 shows T cell margination (CD8+, FIG. 75C) and peripheral IL-6 (FIG. 75A) and MCP-1 (FIG. 75B) concentrations after the first dose and repeat or target doses.

[0250] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in the practice of the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of the claims, and equivalents thereof, be covered thereby.

[0251] [Table 23-1]

[0252] [Table 23-2]

[0253] [Table 23-3]

[0254] [Table 23-4]

[0255]

Table 23-5

[0256]

Table 23-6

[0257]

Table 23-7

[0258]

Table 23-8

[0259]

Table 23-9

[0260]

Table 23-10

[0261]

Table 23-11

[0262]

Table 23-12

[0263]

Table 23-13

[0264]

Table 23-14

[0265]

Table 23-15

[0266]

Table 24-1

[0267]

Table 24-2

[0268]

Table 24-3

[0269]

Table 24-4

[0270]

Table 24-5

[0271]

Table 24-6

[0272]

Table 24-7

[0273]

Table 24-8

[0274]

Table 24-9

[0275]

Table 24-10

[0276]

Table 25-1

[0277]

Table 25-2

[0278]

Table 25-3

[0279]

Table 25-4

[0280]

Table 25-5

[0281]

Table 25-6

[0282]

Table 25-7

[0283]

Table 25-8

[0284]

Table 25-9

[0285]

Table 25-10

[0286]

Table 26-1

[0287]

Table 26-2

[0288]

Table 26-3

[0289]

Table 26-4

[0290]

Table 26-5

[0291]

Table 26-6

[0292]

Table 26-7

[0293]

Table 26-8

[0294]

Table 26-9

[0295]

Table 26-10

[0296] [Table 27-1]

[0297] [Table 27-2]

[0298] [Table 28-1]

[0299] [Table 28-2]

[0300] [Table 28-3]

[0301] [Table 29]

[0302] [Table 30-1]

[0303] [Table 30-2]

[0304] [Table 30-3]

[0305] DLL3 protein UniProtKB deposit Q9NYJ7 (sequence number 1885) >sp|Q9NYJ7|DLL3_HUMAN Δ-like protein 3 OS=Homo sapiens OX=9606 GN=DLL3 PE=1 SV=1 MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAA GGPwards EVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGAR CEFPVHPDGASALPAAPPGLRPGDPQRYLLPPALGLLVAAGVAGAALLLVHVRRRGHSQDAGSRLLAGTPEPSVHALPDALNNLRTQEGSGDGPSSSVDWNRPEDVDPQGIYVISAPSIYAREVATPLFPPLHTGRAGQRQHLLFPYPSSILSVK 51X5 (sequence number 1886) EVQLVESGGGLVQPGGSLTLSCAASLSSVSVLSIAWYRQAPGKKRELVAGISTDGSTVYIDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCYAYSWTTSLPYWGQGTLVTVSS 51X5 CDR1 (SEQ ID NO: 1887) LSSVSVLSIA 51X5 CDR2 (SEQ ID NO: 1888) GISTDGSTVYIDSVKG 51X5 CDR3 (SEQ ID NO: 1889) YSWTTSLPY >NP_058637.1 Delta-like protein 3 isoform 1 precursor [Homo sapiens] (SEQ ID NO: 1892) MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAA GGPwards EVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGAR CEFPVHPDGASALPAAPPGLRPGDPQRYLLPPALGLLVAAGVAGAALLLVHVRRRGHSQDAGSRLLAGTPEPSVHALPDALNNLRTQEGSGDGPSSSVDWNRPEDVDPQGIYVISAPSIYAREVATPLFPPLHTGRAGQRQHLLFPYPSSILSVK DLL3 protein sequence (SEQ ID NO:1893) RSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYL

Claims

**Claim 1** A method of treating cancer, the method comprising administering to a subject an effective amount of a Delta-like ligand 3 (DLL3)-targeting trispecific protein, wherein the Delta-like ligand 3 (DLL3)-targeting trispecific protein is (a) a first domain (A) that specifically binds to human CD3, (b) a second domain (B) that is a half-life extension domain, (c) a third domain (C) that specifically binds to DLL3 and the DLL3-targeting trispecific protein is administered at a dosage of about 1 μg to about 100 mg, a method of treating cancer.

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