CN118159554A

CN118159554A - DLL3 targeted trispecific proteins and methods of use thereof

Info

Publication number: CN118159554A
Application number: CN202280054610.2A
Authority: CN
Inventors: 霍尔格·韦舍; 孙丽萍
Original assignee: Harpoon Therapeutics Inc
Current assignee: Harpoon Therapeutics Inc
Priority date: 2021-06-03
Filing date: 2022-06-02
Publication date: 2024-06-07

Abstract

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

Description

DLL3 targeted trispecific proteins and methods of use thereof

Cross reference

The present application claims the benefit of U.S. provisional patent application No. 63/196,619 filed on 3 month 6 2021, U.S. provisional patent application No. 63/288,939 filed on 13 month 12 2021, and U.S. provisional patent application No. 63/345,150 filed on 24 month 5 2022, each of which are incorporated herein by reference in their entirety.

Background

Selective destruction of individual cells or specific cell types is often required in various clinical settings. For example, specific destruction of tumor cells, while leaving healthy cells and tissues intact, is a major goal of cancer therapies. One such method is to attack immune effector cells such as Natural Killer (NK) cells or Cytotoxic T Lymphocytes (CTLs) and destroy tumor cells by inducing an immune response against the tumor.

Disclosure of Invention

Described herein is a method of treating cancer comprising administering to a subject an effective amount of delta-like ligand 3 (DLL 3) targeted trispecific protein, wherein the protein comprises (a) a first domain (a) that specifically binds human CD3; (b) A second domain (B) which is a half-life extending domain; and (C) a third domain (C) that specifically binds DLL3, wherein the DLL3 targets a trispecific protein to be administered at a dose of about 1 μg to about 100 mg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 1 μg to about 14 mg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 1 μg to about 5 mg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 1 μg to about 2 mg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 1 μg to about 1 mg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 15 μg to about 3600 μg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 15 μg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 45 μg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 135 μg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 405 μg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 1215 μg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 3600 μg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 5 mg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 7 mg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 10 mg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 12 mg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 14 mg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 20 mg. In some embodiments, the DLL3 targeted trispecific protein is administered at a dose of about 50 mg. In some embodiments, the DLL3 targeted trispecific protein is administered once per week. In some embodiments, the DLL3 targeted trispecific protein is administered twice a week. In some embodiments, the DLL3 targeted trispecific protein is administered every other week. In some embodiments, the DLL3 targeted trispecific protein is administered every three weeks. In some embodiments, the DLL3 targets the tri-specific protein for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

Described herein is a method of treating cancer comprising administering to a subject an effective amount of a DLL3 targeted trispecific protein, wherein the protein comprises (a) a first domain (a) that specifically binds human CD3; (b) A second domain (B) which is a half-life extending domain; and (C) a third domain (C) that specifically binds DLL3, wherein the domains are linked in the order of H ₂ N- (a) - (B) - (C) -COOH, or are linked by linkers L1 and L2, and wherein the DLL3 targets the administration of a trispecific protein according to a schedule comprising the steps of: (i) Administering a first dose of DLL3 targeted trispecific protein, and (ii) administering a second dose of DLL3 targeted trispecific protein, wherein the second dose is higher than the first dose. In some embodiments, the first dose is from about 1mg to about 100mg. In some embodiments, the first dose is from about 1mg to about 50mg. In some embodiments, the first dose is from about 1mg to about 20mg. In some embodiments, the first dose is from about 1mg to about 10mg. In some embodiments, the first dose is about 1mg to about 5mg. In some embodiments, the first dose is about 1mg to about 3mg. 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 daily. In some embodiments, the first dose is administered twice daily. In some embodiments, the first dose is administered three times per day. In some embodiments, the first dose is administered five times per 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 every other week. In some embodiments, the first dose is administered 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 from about 1mg to about 100mg. In some embodiments, the second dose is about 1mg to about 50mg. In some embodiments, the second dose is about 50mg to about 100mg. In some embodiments, the second dose is about 7.2mg. In some embodiments, the second dose is about 12mg. In some embodiments, the second dose is about 24mg. In some embodiments, the second dose is about 36mg. 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 daily. In some embodiments, the second dose is administered twice daily. In some embodiments, the second dose is administered three times per day. In some embodiments, the second dose is administered five times per 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 every other week. In some embodiments, the second dose is administered every three weeks. In some embodiments, after administration of the first dose, the second dose is maintained until the end of the schedule. In some embodiments, the second dose is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally.

In some embodiments, the DLL3 targeted 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 a human domain, a humanized domain, an affinity maturation domain, or a combination thereof. In some embodiments, the third domain comprises one or more sequences selected from SEQ ID NO. 1-SEQ ID NO. 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 a humanized domain or a human domain. 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, the linkers L1 and L2 are each independently selected from (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 H ₂ N- (C) -L1- (B) -L2- (a) -COOH. In some embodiments, the DLL3 targets less than about 80kDa of the trispecific protein. In some embodiments, the DLL3 targeted trispecific protein is about 50 to about 75kDa. In some embodiments, the DLL3 targets less than about 60kDa of the trispecific protein. In some embodiments, the DLL3 targeted trispecific protein comprises a sequence selected from the group consisting of SEQ ID NO:1890-SEQ ID NO: 1891. In some embodiments, the DLL3 targeted trispecific protein comprises the sequence as 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 DLL 3. In some embodiments, the cancer is neuroendocrine, prostate, lung, gastric, squamous cell, pancreatic, cholangiocarcinoma, triple negative breast or ovarian cancer. In some embodiments, the cancer is small cell lung cancer. In some embodiments, the cancer is neuroendocrine prostate cancer.

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.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

Fig. 1 shows various domains of an exemplary DLL3 targeted trispecific protein of the present disclosure.

Figure 2 shows the results of a T cell dependent cytotoxicity (TDCC) assay on DMS-153 cells using exemplary DLL3 targeted trispecific proteins containing DLL3 binding domains DH18, DH11, DH67 and DH56 of the present disclosure.

Figure 3 shows the results of TDCC assays on DMS-153 cells using exemplary DLL 3-targeted trispecific proteins containing exemplary DLL3 binding domains DH2, DH43, DH10 and DH6 of the present disclosure.

Figure 4 shows the results of TDCC assays on DMS-153 cells using exemplary DLL 3-targeted trispecific proteins containing exemplary DLL3 binding domains DH82, DH23, DH89 and DH17 of the present disclosure.

Figure 5 shows the results of TDCC assays on DMS-153 cells using exemplary DLL 3-targeted trispecific proteins containing the exemplary DLL3 binding domains DH83, DH12, DH61 and DH29 of the present disclosure.

Figure 6 shows the results of TDCC assays on DMS-153 cells using exemplary DLL3 targeted trispecific proteins containing exemplary DLL3 binding domains DH58 and DH70 of the present disclosure and control trispecific proteins.

Figure 7 shows the results of TDCC assays on DMS-153 cells using exemplary affinity-matured DLL 3-targeted trispecific proteins containing exemplary DLL 3-targeting domains 1a011, 2E05, 1H012, 2E02, and 1C03 of the present disclosure.

Figure 8 shows the results of TDCC assays on DMS-153 cells that bind to trispecific proteins using exemplary affinity-matured DLL3 containing exemplary DLL3 targeting domains 2E010, 2E01, 2H02, 2a04, and 2F11 of the present disclosure.

Figure 9 shows the results of TDCC assays on DMS-153 cells that bind to trispecific proteins using exemplary affinity-matured DLL3 containing the exemplary DLL3 targeting domains 2E011, 3C04, 4H011 and 4D09 of the present disclosure.

Figure 10 shows the results of TDCC assays on DMS-153 cells that bind to trispecific proteins using exemplary affinity-matured DLL3 containing the exemplary DLL3 targeting domains 4B07, 4E02, 4C06, 3H011, and 3D07 of the present disclosure.

Figure 11 shows the results of TDCC assays on DMS-153 cells using exemplary affinity-matured DLL 3-targeted trispecific proteins containing exemplary DLL3 binding domains 3H06 and 4B011 of the present disclosure and parent DLL conjugate domains DH43, DH6, as well as control trispecific proteins.

FIG. 12 shows the results of TDCC assays on DMS-153 cells using exemplary purified affinity matured CHO-expressed DLL3 binding trispecific proteins containing exemplary DLL3 targeting domains 2E05-M106Y, 2E05-M106Q, 4D09-M34L and 4H11-M34L of the present disclosure.

Figure 13 shows the results of TDCC assays on DMS-153 cells using exemplary purified affinity matured CHO-expressed DLL 3-targeted trispecific proteins containing exemplary DLL3 binding domains 1a011 (labeled 1a11 in figure 13), 1H012 (labeled 1H12 in figure 13), 2E02 and 2E05 of the present disclosure.

Figure 14 shows the results of TDCC assays on DMS-153 cells using exemplary purified affinity matured CHO-expressed DLL 3-targeted trispecific proteins containing exemplary DLL3 binding domains 2H02, 3C04, 4D09 and 4H11 of the present disclosure.

Figure 15 shows the results of TDCC assays on DMS-153 cells using exemplary purified DLL 3-targeted trispecific proteins containing parent exemplary DLL3 binding domains DH43 and DH6 and GFP-targeted control trispecific proteins.

Figure 16 shows the results of TDCC assays on DMS-153 cells using an exemplary DLL3 targeting trispecific protein containing an exemplary DLL3 binding domain of the present disclosure from a second round of affinity maturation.

FIG. 17 shows an image of 10-20% TRIS glycine SDS-PAGE loaded with 2.4. Mu.g of non-reducing protein and stained with Coomassie per lane. Lane numbers are indicated by numbers at the top of the gel image and migration of molecular weight standards is indicated by numbers on the right side of the gel image (in kilodaltons). Gel loading: lane 1, empty; lane 2, molecular weight standard; lane 3, empty; lane 4, anti-DLL 3 trispecific protein containing DLL3 binding domain 51G 2; lane 5, anti-DLL 3 trispecific protein containing DLL3 binding domain 51G 10; lane 6, anti-DLL 3 trispecific protein containing DLL3 binding domain 51H 5; lane 7, anti-DLL 3 trispecific protein containing DLL3 binding domain 51X 5; lane 8, anti-DLL 3 trispecific protein containing DLL3 binding domain 52B 1; lane 9, anti-DLL 3 trispecific protein containing DLL3 binding domain 52C 4; lane 10, anti-DLL 3 trispecific protein containing DLL3 binding domain 52D 4; lane 11, anti-DLL 3 trispecific protein containing DLL3 binding domain 51 A2; lane 12, anti-DLL 3 trispecific protein containing DLL3 binding domain 51 A5; lane 13, anti-DLL 3 trispecific protein containing DLL3 binding domain 51F 3; lane 14, empty; and lane 15, empty.

Figure 18 shows the results of TDCC assays on DMS-53 cells using exemplary purified affinity matured CHO-expressed DLL 3-targeted trispecific proteins containing exemplary DLL3 binding domains 51G2, 51G10, 51H5, 51X5, 52B1, 52C4, 52D4, 51A2 of the present disclosure and parent DLL3 conjugate domain DH6 as well as control trispecific proteins.

Figure 19 shows the results of TDCC assays on DMS-153 cells using exemplary purified affinity matured CHO-expressed DLL3 targeted trispecific proteins containing exemplary DLL3 binding domains 51G2, 51G10, 51H5, 51X5, 52B1, 52C4, 52D4, 51A2 of the present disclosure and parent DLL3 binding domain DH6 and GFP-targeted controls binding trispecific proteins.

FIG. 20 provides a schematic of a DLL3 targeted trispecific protein containing an exemplary DLL3 binding protein (DLL 3 conjugate), CD3 binding domain (anti-CD 3 εscFv) and albumin binding (anti-ALB) domain of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 orientation (TAC orientation).

Figure 21 provides a schematic of a DLL3 targeted trispecific protein containing an exemplary DLL3 binding protein (DLL 3 conjugate), CD3 binding domain (anti-CD 3 epsilon scFv) and albumin binding (anti-ALB) domain of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 orientation (CAT orientation).

FIG. 22 shows the results of T cell dependent cytotoxicity (TDCC) assays on NCI-H2171 cells using exemplary DLL3 trispecific proteins containing the DLL3 binding domain 52D04 of the present disclosure in the presence of Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration or in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration.

Figure 23 shows the results of a T cell dependent cytotoxicity (TDCC) assay on DMS-79 cells using an exemplary DLL 3-targeted trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration or in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, tested in the presence or absence of Human Serum Albumin (HSA).

Fig. 24 shows the results of T cell dependent cytotoxicity (TDCC) assays on SHP77 cells using exemplary DLL3 trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in the presence of Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration or in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration.

Fig. 25 shows the results of T cell dependent cytotoxicity (TDCC) assays on WM2664 cells using exemplary DLL3 trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in the presence of Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration or in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration.

FIG. 26 depicts a comparison of binding of an exemplary DLL 3-targeted trispecific protein containing the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration to human T cells from four different donors, as compared to the binding of cells with a control of secondary antibody alone or without any antibody or trispecific molecule.

Figure 27 depicts a comparison of binding of an exemplary DLL3 targeted trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration to human T cells from four different donors versus cells with a control of a secondary antibody alone or without any antibodies or trispecific molecules.

FIG. 28 depicts a comparison of binding of an exemplary DLL 3-targeted trispecific protein containing the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration to a human DLL3 expressing cell line NCI-H82 (upper left), SHP77 (upper right), DMS53 (lower left) or NCI-H2171 (lower right) with a trispecific molecule having a GFP binding domain.

FIG. 29 depicts a comparison of binding of an exemplary DLL 3-targeted trispecific protein containing the DLL3 binding domain 52D04 of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration to a human DLL3 expressing cell line NCI-H82 (upper left), SHP77 (upper right), DMS53 (lower left) or NCI-H2171 (lower right) with a trispecific molecule having a GFP binding domain.

Figure 30 shows the results of TDCC assays on NCI-H82 cells using exemplary DLL 3-targeted trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Fig. 31 shows the results of TDCC assays on SHP77 cells using exemplary DLL 3-targeted trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Figure 32 shows the results of TDCC assays on DMS53 cells using exemplary DLL 3-targeted trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Figure 33 shows the results of TDCC assays on NCI-H2171 cells using an exemplary DLL 3-targeted trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Figure 34 shows the results of TDCC assays on NCI-H82 cells using exemplary DLL 3-targeted trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in the anti-CD 3-anti-ALB-anti-DLL 3 (CAT) configuration, tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Fig. 35 shows the results of TDCC assays on SHP77 cells using exemplary DLL 3-targeted trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in the anti-CD 3-anti-ALB-anti-DLL 3 (CAT) configuration, tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Figure 36 shows the results of TDCC assays on DMS53 cells using exemplary DLL 3-targeted trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in the anti-CD 3-anti-ALB-anti-DLL 3 (CAT) configuration, tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Figure 37 shows the results of TDCC assays on NCI-H2171 cells using an exemplary DLL 3-targeted trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration, tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

FIG. 38 shows the results of flow cytometry measurements of CD69 expression on T cells co-cultured with NCI-H82 cells with a titration of an exemplary DLL 3-targeted trispecific protein containing the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

FIG. 39 shows the results of flow cytometry measurements of CD25 expression on T cells co-cultured with NCI-H82 cells with a titration of an exemplary DLL 3-targeted trispecific protein containing the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Figure 40 shows the results of flow cytometry measurements of CD69 expression on T cells co-cultured with DMS53 cells with a titration of an exemplary DLL 3-targeted trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Fig. 41 shows the results of flow cytometry measurements of CD25 expression on T cells co-cultured with DMS53 cells with a titration of an exemplary DLL 3-targeted trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, the assay tested in the presence of Human Serum Albumin (HSA).

FIG. 42 shows the results of flow cytometry measurements of CD69 expression on T cells co-cultured with NCI-H82 cells with a titration of an exemplary DLL 3-targeted trispecific protein containing the DLL3 binding domain 52D04 of the present disclosure in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

FIG. 43 shows the results of flow cytometry measurements of CD25 expression on T cells co-cultured with NCI-H82 cells with a titration of an exemplary DLL 3-targeted trispecific protein containing the DLL3 binding domain 52D04 of the present disclosure in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration, tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Figure 44 shows the results of flow cytometry measurements of CD69 expression on T cells co-cultured with DMS53 cells with a titration of an exemplary DLL 3-targeted trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Figure 45 shows the results of flow cytometry measurements of CD25 expression on T cells co-cultured with DMS53 cells with a titration of an exemplary DLL 3-targeted trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration, the assay tested in the presence of Human Serum Albumin (HSA).

FIG. 46 shows the results of IFNγ measurements in conditioned medium from co-cultures of T cells and NCI-H82 cells incubated with a titration of an exemplary DLL3 targeting trispecific protein containing the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, the measurements being tested in the presence of Human Serum Albumin (HSA).

Fig. 47 shows the results of ifnγ measurements in conditioned medium from co-cultures of T cells and SHP77 cells incubated with a titration of an exemplary DLL 3-targeting trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA).

FIG. 48 shows the results of IL-2 measurements in conditioned medium from co-cultures of T cells and NCI-H82 cells incubated with a titration of an exemplary DLL3 targeting trispecific protein containing the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, the measurements being tested in the presence of Human Serum Albumin (HSA).

Fig. 49 shows the results of IL-2 measurements in conditioned medium from co-cultures of T cells and SHP77 cells incubated with a titration of an exemplary DLL 3-targeting trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA).

Figure 50 shows the results of tnfα measurements in conditioned medium from co-cultures of T cells and NCI-H82 cells incubated with a titration of an exemplary DLL 3-targeting trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA).

Fig. 51 shows the results of tnfα measurements in conditioned medium from co-cultures of T cells and SHP77 cells incubated with a titration of an exemplary DLL 3-targeting trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA).

Figure 52 shows the results of ifnγ measurements in conditioned medium from co-cultures of T cells and NCI-H82 cells incubated with a titration of an exemplary DLL 3-targeting trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA).

Figure 53 shows the results of ifnγ measurements in conditioned medium from co-cultures of T cells and SHP77 cells incubated with a titration of an exemplary DLL3 targeting trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA).

Figure 54 shows the results of IL-2 measurements in conditioned medium from co-cultures of T cells and NCI-H82 cells incubated with a titration of an exemplary DLL 3-targeting trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA).

Figure 55 shows the results of IL-2 measurements in conditioned medium from co-cultures of T cells and SHP77 cells incubated with a titration of an exemplary DLL3 targeting trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA).

Figure 56 shows the results of tnfα measurements in conditioned medium from co-cultures of T cells and NCI-H82 cells incubated with a titration of an exemplary DLL3 targeting trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA).

Fig. 57 shows the results of tnfα measurements in conditioned medium from co-cultures of T cells and SHP77 cells incubated with a titration of an exemplary DLL3 targeting trispecific protein containing DLL3 binding domain 52D04 of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration, the measurements were tested in the presence of Human Serum Albumin (HSA).

FIG. 58 depicts that exemplary DLL 3-targeted trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in either an anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration or an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration are capable of inhibiting tumor growth in mice injected with a mixture of human T cells and NCI-H82 small cell lung cancer cells at a dose of 20 μg/kg, 100 μg/kg or 500 μg/kg.

FIG. 59 depicts the growth of NCI-H82 xenograft tumors in mice injected with human T cells that were able to be eliminated at doses of 10 μg/kg and 100 μg/kg with an exemplary DLL 3-targeted trispecific protein containing the DLL3 binding domain 52D04 of the present disclosure in an anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration.

FIG. 60 depicts that exemplary DLL 3-targeted trispecific proteins containing the DLL3 binding domain 52D04 of the present disclosure in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration are capable of inhibiting 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.

Fig. 61 depicts the pharmacokinetic profile of exemplary DLL 3-targeted trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in either the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration (ID numbers 1 and 2) or anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration (ID numbers 3 and 4). Serum levels of DLL3 targeted trispecific proteins at various time points after injection into the cynomolgus monkey at 0.3mg/kg are shown in the graph.

Figure 62 depicts pharmacokinetic profiles of exemplary DLL3 targeted trispecific proteins containing DLL3 binding domains 52D04 of the present disclosure in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration at various time points after injection into a cynomolgus monkey at 1mg/kg or 10mg/kg are shown in the graph.

FIG. 63 depicts a transient cytokine increase after binding TriTAC molecules or vehicle controls at 1mg/kg and 10mg/kg of the first administration of an exemplary DLL3 of the present disclosure. The upper panel shows a transient increase in ifnγ, the second panel shows a transient increase in IL-6, and the third panel shows a transient increase in IL-10.

Figure 64 shows the results of TDCC assays on DMS53 cells using exemplary DLL 3-targeted trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration, measured in the presence of 8.4% cynomolgus monkey serum using freshly thawed proteins, or using proteins present in serum samples from cynomolgus monkeys collected 168h after administration of 10mg/kg of DLL 3-targeted trispecific proteins.

FIG. 65 shows the phase 1/2 assay design for a DLL3 trispecific antigen binding protein.

Figure 66 shows the time of treatment for the patient, the weekly dose per patient, the number of previous therapies, and the patient identification number.

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

Fig. 68 shows the decrease in target lesions in patients over time.

Fig. 69 shows pharmacokinetic data for DLL3 trispecific antigen-binding proteins from different drug administration queues.

Fig. 70 shows the results of the flow analysis. Figure 70A shows T cell edge levels after treatment. Figure 70B shows T cell activation marker induction after treatment.

Fig. 71A shows the change in target lesion diameter of patient 111 over time. The BCT scan of fig. 71 shows a reduction in the sum of target lesion diameters for patient 111.

Fig. 72A shows the change in target lesion diameter of patient 112 over time. The BCT scan of fig. 72 shows a reduction in the sum of target lesion diameters for patient 112.

Fig. 73 shows the change in target lesion diameter of patient 113 over time.

Figure 74 shows a concentration-time profile (figure 74A) and Cmax by dosimeter (figure 74B).

FIG. 75 shows T cell edge set (CD8+, FIG. 75C) and peripheral IL-6 (FIG. 75A) and MCP-1 (FIG. 75B) concentrations after the first and repeated or target doses.

Detailed Description

In some embodiments, described herein are proteins that specifically bind delta-like ligand 3 (DLL 3) and multispecific (e.g., trispecific) proteins containing the same, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for making such proteins. Also provided are methods of using at least one of the following in the prevention and/or treatment of diseases, conditions and disorders: the disclosed DLL3 binding proteins or DLL3 containing the same target trispecific proteins. DLL3 targets trispecific proteins that are capable of specifically binding to DLL3 as well as CD3 and have half-life extending domains, such as domains capable of specifically binding to human Albumin (ALB). FIG. 1 depicts one non-limiting example of a trispecific DLL3 binding protein. In some embodiments, the DLL3 targeted trispecific protein comprises an antibody, e.g., a trispecific antibody.

Certain definitions

"Antibody" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Human light chains comprise a variable domain (VL) and a constant domain (CL), wherein the constant domain can be easily classified as either kappa or lambda based on amino acid sequence and locus. Each heavy chain comprises a variable domain (VH) and a constant region comprising three domains, called CH1, CH2 and CH3 in the case of IgG, igA and IgD (IgM and IgE have the fourth domain CH 4). In the IgG, igA and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a proline and cysteine rich segment of variable length (typically about 10 to about 60 amino acids in IgG). The variable domains in both the light and heavy chains are linked to the constant domain by a "J" region of about 12 or more amino acids, and the heavy chain also has a "D" region of about 10 additional amino acids. Each class of antibodies also contains interchain and intrachain disulfide bonds formed by pairs of cysteine residues. There are two types of natural disulfide bridges or disulfide bonds in immunoglobulin molecules: inter-chain disulfide bonds and intra-chain disulfide bonds. The positions and numbers of interchain disulfide chains vary depending on immunoglobulin class and species. The interchain disulfide bonds are located on the surface of the immunoglobulin, are readily accessible to solvents, and are generally relatively easily reduced. There are four interchain disulfide bonds in the human IgG1 isotype, one interchain disulfide bond for each heavy to light chain, and two interchain disulfide bonds between the heavy chains. Chain association does not require interchain disulfide bonds. It is well known that the cysteine-rich IgG1 hinge region of a heavy chain is generally composed of three parts: an upper hinge, a core hinge, and a lower hinge. Those skilled in the art will appreciate that the IgG1 hinge region contains cysteines in the heavy chain, which contain interchain disulfide bonds (two heavy/heavy, two heavy/light), which provides structural flexibility to facilitate Fab movement. The inter-chain 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 bond between the heavy chains is at positions C226 and C229 (all numbered according to the EU index according to Kabat et al, infra).

As used herein, the term "antibody" includes polyclonal antibodies (polyclonal antibodies), polyclonal antibodies (multiclonal antibodies), monoclonal antibodies, chimeric antibodies, humanized and primatized (CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intracellular antibodies (intrabodies), multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), immunospecific antibody fragments such as Fd, fab, F (ab ') 2, F (ab') fragments, single chain fragments (e.g., scFv and ScFvFc), disulfide-linked Fv (sdFv), fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VH, VL or VHH domains); and derivatives thereof, including Fc fusions and other modifications, as well as any other immunoreactive molecules, so long as they comprise a domain having a binding site for preferentially associating or binding a DLL3 protein. Furthermore, unless the context limits dictate otherwise, the term also includes all classes of antibodies (i.e., igA, igD, igE, igG and IgM) and all subclasses (i.e., igG1, igG2, igG3, igG4, igA1, and IgA 2). The heavy chain constant domains corresponding to different classes of antibodies are generally represented by the corresponding lowercase letters α, δ, ε, γ and μ, respectively. The light chain of an antibody from any vertebrate species can be assigned to one of two distinct types (called kappa and lambda) based on the amino acid sequence of its constant domain.

In some embodiments, the DLL3 binding domain of the DLL3 targeted trispecific proteins of the present disclosure comprises an antibody that is heavy chain only, such as a VH or VHH domain. In some cases, the DLL3 binding protein comprises an antibody that is only a heavy chain of an engineered human VH domain. In some examples, the engineered human VH domain is generated by panning a phage display library. In some embodiments, the DLL3 binding domain of the DLL3 targeting trispecific proteins of the present disclosure comprises a VHH. As used herein, the term "VHH" refers to a single chain antibody binding domain that lacks a light chain. In some cases, the VHH is derived from a naturally light chain-free antibody type found in camelidae or cartilaginous fish, or from a synthetic and non-immune VHH that can be constructed accordingly. Each heavy chain comprises a variable region encoded by a V exon, a D exon, and a J exon. In some cases, the VHH is a native VHH such as a camelid-derived VHH, or a recombinant protein comprising a heavy chain variable domain. In some embodiments, the VHH is derived from a species selected from camel, llama, camel, dromedary, and cartilaginous fish (such as, but not limited to, shark). In another embodiment, the VHH is derived from a alpaca (such as, but not limited to Hua Ka about alpaca or threo camel).

As used herein, "variable region" or "variable domain" refers to portions of the variable domain that vary greatly in sequence between antibodies and are used for binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed across the variable domains of antibodies. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light chain (VL) and heavy chain (VH) variable domains. The more highly conserved parts of the variable domains are called the Framework (FR). The variable domains of the natural heavy and light chains each comprise four FR regions connected by three CDRs, which predominantly adopt the β -sheet configuration, the CDRs forming loops that connect the β -sheet structure and in some cases form part of the β -sheet structure. The CDRs in 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 the antibody (see Kabat et al Sequences of Immunological Interest, fifth edition, national Institute of Health, bethesda, md. (1991)). Although the constant domains are not directly involved in binding of antibodies to antigens, various effector functions are shown, such as antibody involvement in antibody-dependent cellular cytotoxicity. scFv fragments (for single chain variable fragments) are in some cases obtained by genetic engineering, associated with VH and VL regions of antibodies in a single polypeptide chain, separated by peptide linkers.

In some embodiments of the disclosure, the DLL3 binding domain, e.g., the DLL3 binding domain of a DLL3 targeted trispecific protein, comprises a single domain antibody, e.g., a heavy chain-only antibody, e.g., a VH or VHH domain, and comprises three CDRs. In some embodiments, such heavy chain-only antibodies bind DLL3 in monomeric form, which does not rely on dimerization with VL (light chain variable) regions to obtain optimal binding affinity. In some embodiments of the disclosure, the CD3 binding domain of the DLL3 targeted trispecific protein comprises an scFv. In some embodiments of the disclosure, the albumin binding domain of the DLL3 targeted trispecific protein comprises a heavy chain-only antibody, e.g., a single domain antibody comprising a VH domain or a VHH domain.

In some embodiments, unless otherwise indicated, the assignment of amino acids to each domain, framework region, and CDR is according to one of the numbering schemes provided in the following documents: kabat et al, (1991) Sequences of Proteins of Immunological Interest (5 th edition), USDept.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, code (2007) Handbook of Therapeutic Antibodies, 3 rd edition, wily-VCH VERLAG GmbH and Co or AbM (Oxford Molecular/MSIPharmacopia). The CDRs of the present disclosure do not necessarily correspond to the Kabat numbering convention. In some embodiments of the disclosure, the DLL3 binding protein comprises a single domain antibody, e.g., a heavy chain-only antibody, e.g., a VH or VHH domain, and comprises three CDRs. In some embodiments, such heavy chain-only antibodies bind DLL3 in monomeric form, which does not rely on dimerization with VL (light chain variable) regions to obtain optimal binding affinity.

"Variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variants thereof refer to the numbering system of the heavy chain variable domain or the light chain variable domain for antibody assembly (compilation) in Kabat et al Sequences of Proteins of Immunological Interest, public HEALTH SERVICE, national Institutes of Health, bethesda, md. (1991). Using such numbering system, the actual linear amino acid sequence may contain fewer or additional shortened or inserted amino acids corresponding to the FR or CDR of the variable domain. For example, the heavy chain variable domain can include a single amino acid insertion (residue 52a, according to Kabat) following residue 52 of H2 and an insertion residue (e.g., residues 82a, 82b, and 82c, etc., according to Kabat) following heavy chain FR residue 82. The Kabat numbering of residues of a given antibody can be determined by alignment of the antibody sequences at regions of homology with the "standard" Kabat numbering sequences.

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

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

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, code 6 th edition 1980). Briefly, the term is intended to encompass quantitative measures of drug elimination time course. The elimination of most drugs is exponential (i.e., follows a first order kinetics) because drug concentration is generally not close to that required for saturation of the elimination process. The rate of an exponential process can be expressed in terms of its rate constant k, which represents the proportional change per unit time, or in terms of its half-life t1/2, i.e. the time required to complete a 50% process. The units of these two constants are time-1 and time, respectively. The first order rate constant is only related to the half-life of the reaction (kχt1/2=0.693) and can be interchanged accordingly. Since the first order elimination kinetics indicates a constant drug fraction lost per unit time, the log plot of drug concentration versus time is linear at all times after the initial distribution phase (i.e., after drug absorption and distribution is complete). From such a graph, the half-life of drug elimination can be accurately determined.

As used herein, the term "binding affinity" refers to the affinity of a protein described in the present disclosure for its binding to a target, and is expressed numerically using a "Kd" value. If two or more proteins are indicated to have comparable binding affinities for their binding targets, the Kd values for each protein to bind to its binding target are within ±2 times of each other. If two or more proteins are indicated to have comparable binding affinities for a single binding target, the Kd values for each protein to bind to the single binding target are within ±2-fold of each other. If a protein is indicated to bind to two or more targets with comparable binding affinities, the Kd values for the protein to bind to the two or more targets are within ±2 times of each other. In general, higher Kd values correspond to weaker binding. In some embodiments, "Kd" is measured by a radiolabeled antigen binding assay (RIA) or a surface plasmon resonance assay using BIAcore ^TM -2000 or BIAcore ^TM -3000 (BIAcore, inc., piscataway, n.j.). In certain embodiments, the "binding rate (on-rate)" or "association rate (rate of association)" or "association rate" or "kon" and the "off-rate" or "rate of dissociation (rate of dissociation)" or "dissociation rate (dissociation rate)" or "koff) are also determined using surface plasmon resonance techniques of BIAcore ^TM -2000 or BIAcore ^TM -3000 (BIAcore, inc., piscataway, N.J.). In a further embodiment, use is made ofSystems (Pall Life Sciences) measures "Kd", "kon" and "koff". In use/>In an exemplary method for systematically measuring binding affinity, a ligand, such as biotinylated human or cynomolgus monkey DLL3, is immobilized at/>The streptavidin capillary sensor tip surface was then activated with about 20-50 μg/ml human or cynomolgus monkey DLL3 protein according to manufacturer's instructions. PBS/casein solution was also introduced as blocking agent. For association kinetics measurements, the DLL3 binding protein variant is introduced at a concentration of about 10ng/mL to about 100 μg/mL, about 50ng/mL to about 5 μg/mL, or about 2ng/mL to about 20 μg/mL. In some embodiments, DLL3 binding single domain protein in about 2ng/mL to about 20 u g/mL concentration using. Complete dissociation was observed in the negative control, assay buffer without binding protein. The kinetic parameters of the binding reaction are then determined using appropriate tools (e.g., forteBio software).

One embodiment provides a DLL3 binding protein comprising a single domain antibody (also referred to herein as a DLL3 binding domain, such as the DLL3 binding domain of a DLL3 trispecific antibody of the present disclosure) comprising a CDR1 sequence selected from the sequences of SEQ ID nos. 443-884 and 1887, a CDR2 sequence comprising the sequences selected from the sequences of SEQ ID nos. 885-1326 and 1888, and a CDR3 sequence comprising the sequences selected from the sequences of SEQ ID nos. 1327-1768 and 1889. It is contemplated that in some embodiments, the DLL3 binding proteins of the present disclosure are relatively small and in some embodiments no more than 25kD, no more than 20kDa, no more than 15kDa, or no more than 10kDa. In some cases, if the EGFR-binding protein is a peptide or small molecule entity, it is 5kDa or less.

In one aspect, the DLL3 targeted trispecific protein (also referred to herein as DLL3 binding trispecific protein, DLL3 trispecific protein, or DLL3 TriTAC ^TM) comprises: (a) a first domain (a) that specifically binds human CD3; (b) A second domain (B) which is a half-life extending domain; and (C) a third domain (C) that specifically binds DLL3.DLL3 targets three domains in a trispecific protein in any order. Thus, the domain order of DLL3 targeting trispecific proteins is assumed to be:

H₂N-(A)-(B)-(C)-COOH，

H₂N-(A)-(C)-(B)-COOH，

H₂N-(B)-(A)-(C)-COOH，

H₂N-(B)-(C)-(A)-COOH，

H ₂ N- (C) - (B) - (A) -COOH or

H₂N-(C)-(A)-(B)-COOH。

In some embodiments, the domain order of DLL3 targeting trispecific proteins is H ₂ N- (a) - (B) - (C) -COOH. In some embodiments, the domain order of DLL3 targeting trispecific proteins is H ₂ N- (a) - (C) - (B) -COOH. In some embodiments, the domain order of DLL3 targeting trispecific proteins is H ₂ N- (B) - (a) - (C) -COOH. In some embodiments, the domain order of DLL3 targeting trispecific proteins is H ₂ N- (B) - (C) - (a) -COOH. In some embodiments, the domain order of DLL3 targeting trispecific proteins is H ₂ N- (C) - (B) - (a) -COOH. In some embodiments, the domain order of DLL3 targeting trispecific proteins is H ₂ N- (C) - (a) - (B) -COOH.

In some embodiments, the DLL 3-targeted trispecific protein has the HSA (also referred to herein) binding domain as an intermediate domain such that the domain order is H ₂ N- (a) - (B) - (C) -COOH or H ₂ N- (C) - (B) - (a) -COOH. It is contemplated that in such embodiments where the HSA binding domain is an intermediate domain, the CD3 and DLL3 binding domains are provided with additional flexibility to bind to their respective targets.

In some embodiments, the trispecific binding protein comprises a third domain that specifically binds DLL3, which in some cases is a DLL3 binding single domain antibody that binds DLL3 with an affinity comparable to or better than the reference DLL3 binding parent molecule. In some embodiments, the third domain comprises an affinity-matured DLL3 binding molecule (e.g., affinity-matured DLL3 binding single domain antibody), and is derived from a DLL3 binding parent molecule that comprises 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 excellent stability relative to the selected destabilizing agent, such as stability of reference DLL3 binding to the parent molecule. In some embodiments, affinity-matured DLL3 binding molecules are identified in a process that includes panning for DLL3 proteins (such as human DLL3 proteins) for one or more pre-candidate DLL3 binding molecules derived from one or more DLL3 binding parent molecules expressed in a phage display library. In some embodiments, the pre-candidate DLL3 binding molecule comprises amino acid substitutions in variable regions, CDRs, or framework residues relative to the parent molecule.

As used herein, "phage display" refers to a technique of displaying a variant polypeptide as a fusion protein with at least a portion of a coat protein on the surface of a phage, filamentous phage, particle. Phage display has utility in that large randomized libraries of protein variants can be quickly and efficiently selected for those sequences that bind to target molecules with high affinity. Libraries of peptides and proteins displayed on phage have been used to screen millions of polypeptides with specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins by fusion with gene III or gene VIII of filamentous phage. Wells and Lowman, curr.Opin. Structure. Biol,3:355-362 (1992) and references cited therein. In monovalent phage display, a library of proteins or peptides 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 phage particles display one copy of the fusion protein or no fusion protein. The avidity effect is reduced relative to multivalent phage, such that selection is based on intrinsic ligand affinity, and phagemid vectors are used, thus simplifying DNA manipulation. Lowman and Wells, methods: A companion to Methods in Enzymology,3:205-0216 (1991).

In some embodiments, panning includes using different binding times and concentrations to identify DLL3 binding molecules from pre-candidate DLL3 binding molecules that have increased or decreased association rates. In some embodiments, panning includes using different wash times to identify DLL3 binding molecules from pre-candidate DLL3 molecules that have increased or decreased rates of dissociation. In some embodiments, panning involves the use of different binding times and different washing times. In some embodiments, one or more stabilizing mutations are combined to increase the stability of affinity-matured DLL3 binding molecules, e.g., by shuffling to generate second-stage combinatorial libraries of such mutants, and a second round of panning is performed, followed by binding selection.

In some embodiments, affinity for DLL3 proteins (such as human DLL3 proteins) comprised by the affinity-matured DLL3 binding molecule is equivalent to or better 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 or designed to have specific for DLL3 epitopes to which the DLL3 binding parent molecule is specific. In regard to the latter, in some embodiments, affinity-matured DLL3 binding molecules are tested more successfully in animal models if they react with human DLL3 and the corresponding target of animal model mouse DLL3 or cynomolgus monkey DLL3. In some embodiments, the parent DLL3 binding molecule binds human DLL3 with an affinity of about 10nM or less and binds cynomolgus monkey DLL3 with an affinity of about 15nM or less. In some embodiments, the affinity-matured DLL3 binding molecules identified after one round of panning bind human DLL3 with an affinity of about 5nM or less and bind cynomolgus monkey DLL3 with an affinity of about 7.5nM or less. In some embodiments, the affinity-matured DLL3 binding molecules identified after two rounds of panning bind human DLL3 with an affinity of about 2.5nM or less and bind cynomolgus monkey DLL3 with an affinity of about 3.5nM or less.

In some embodiments, domain a, domain B, and domain C of the trispecific binding proteins of the present disclosure are independently antigen specific binding domain polypeptides that specifically bind to a target, such as a target on a diseased cell, or a target on other cells that support a diseased state, such as a target on a stromal cell that supports tumor growth or a target on an immune cell that supports disease-mediated immunosuppression. In some examples, antigen-specific binding domains include antibodies, heavy chain-only antibodies including single chain antibodies, fab, fv, T cell receptor binding domains, ligand binding domains, receptor binding domains, domain antibodies, single domain antibodies, minibodies, nanobodies, peptibodies (peptabodies) or various other antibody mimics (e.g., affimer, affitin, alphabody, atrimer, CTLA 4-based molecules, adnectin, anticalin, kunitz domain-based proteins, avimers, cysteine junctions (knottin), fynomer, darpin, affibodies (affibodies), affilin, monoclonal antibodies, and proteins based on repeated proteins).

In some embodiments, the DLL3 targeted trispecific proteins described herein comprise DLL binding polypeptides 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., third domain (C)) having at least 70% -95% or more homology to a sequence selected from the group consisting of SEQ ID No.1-SEQ ID No.442 and SEQ ID No.1886, subsequences thereof and variants thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3 binding polypeptide (i.e., 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 No.1-SEQ ID No.442 and SEQ ID No.1886, subsequences thereof, and variants thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3 binding polypeptide (i.e., third domain (C)) having at least 70% -95% or more identity to a sequence selected from the group consisting of SEQ ID No.1-SEQ ID No.442 and SEQ ID No.1886, subsequences thereof and variants thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3 binding polypeptide (i.e., third domain (C)) that has 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 No.1-SEQ ID No.442 and SEQ ID No.1886, a subsequence thereof, and variants thereof.

The DLL3 targeting trispecific proteins described herein are designed to achieve specific targeting of DLL3 expressing cells by recruiting cytotoxic T cells. In some embodiments, this increases efficacy compared to ADCC (antibody dependent cell mediated cytotoxicity), which uses full length antibodies against a single antigen and is unable to directly recruit cytotoxic T cells. In contrast, by conjugating specifically expressed CD3 molecules on these cells, DLL 3-targeted trispecific proteins can crosslink cytotoxic T cells with DLL 3-expressing cells in a highly specific manner, leading to the cytotoxic potential of the T cells to the target cells. The DLL 3-targeted trispecific proteins described herein bind to cytotoxic T cells via binding to surface-expressed CD3 proteins (which form part of the TCR). Several DLL3 trispecific antigen-binding proteins bind simultaneously to CD3 and DLL3 expressed on the surface of a particular cell, causing T cell activation and mediating subsequent lysis of the particular DLL3 expressing cell. Thus, DLL 3-targeted trispecific proteins are envisioned to exhibit strong, specific and efficient target cell killing. In some embodiments, a DLL 3-targeted trispecific protein described herein stimulates killing of target cells by cytotoxic T cells to eliminate pathogenic cells (e.g., tumor cells expressing DLL 3). In some such embodiments, the cells are selectively eliminated, thereby reducing the likelihood of toxic side effects.

The DLL 3-targeted trispecific proteins described herein have additional therapeutic advantages over traditional monoclonal antibodies and other smaller bispecific molecules. In general, the effectiveness of recombinant protein drugs depends to a large extent on the intrinsic pharmacokinetics of the protein itself. One such benefit here is that the DLL3 targeted trispecific proteins described herein have an extended pharmacokinetic elimination half-life due to having a half-life extending domain, such as a domain that specifically binds serum albumin (e.g., human serum albumin, HSA). In this aspect, in some embodiments, the DLL3 targeted trispecific proteins described herein have an extended serum elimination half-life of about two days, three days, about five days, about seven days, about 10 days, about 12 days, or about 14 days. This is in contrast to other binding proteins (e.g., biTE or DART molecules), which have a relatively much shorter elimination half-life. For example, biTE CD19 xcd 3 bispecific scFv-scFv fusion molecules require drug delivery by continuous intravenous infusion (iv) due to their short elimination half-life. The longer intrinsic half-life of DLL3 targeted trispecific proteins solves this problem, enabling increased therapeutic potential such as low dose pharmaceutical formulations, reduced periodic administration and/or novel pharmaceutical compositions.

The DLL3 targeted trispecific proteins described herein also have an optimal size for tissue penetration and tissue distribution enhancement. The larger size limits or prevents penetration or distribution of the protein in the target tissue. The DLL3 targeted trispecific proteins described herein avoid this by having a small size that achieves enhanced tissue penetration and distribution. Thus, in some embodiments, the DLL3 targeted trispecific proteins described herein are about 50kDa to about 80kDa, about 50kDa to about 75kDa, about 50kDa to about 70kDa, or about 50kDa to about 65kDa in size. In some embodiments, the DLL3 targets a trispecific protein of a size of less than about 60kDa. Thus, DLL3 targets IgG antibodies of a size better than that of about 150kDa and BiTE and DART diabody molecules of about 55kDa, but they are not half-life and therefore are rapidly cleared by the kidney.

In further embodiments, the DLL3 targeted trispecific proteins described herein have an optimal size for tissue penetration and distribution enhancement. In these embodiments, DLL3 targeted trispecific proteins are constructed to be as small as possible while retaining specificity for their targets. Thus, in these embodiments, the DLL3 targeted trispecific proteins described herein are about 20kDa to about 40kDa in size, or about 25kDa to about 35kDa, to about 40kDa, to about 45kDa, to about 50kDa, to about 55kDa, to about 60kDa, to about 65kDa. In some embodiments, the DLL3 targeted trispecific proteins described herein are about 50kDa, 49kDa, 48kDa, 47kDa, 46kDa, 45kDa, 44kDa, 43kDa, 42kDa, 41kDa, 40kDa, about 39kDa, about 38kDa, about 37kDa, about 36kDa, about 35kDa, about 34kDa, about 33kDa, about 32kDa, about 31kDa, about 30kDa, about 29kDa, about 28kDa, about 27kDa, about 26kDa, about 25kDa, about 24kDa, about 23kDa, about 22kDa, about 21kDa, or about 20kDa in size. An exemplary way to achieve small size is by using single domain antibody (sdAb) fragments for each domain. For example, a particular DLL3 trispecific antigen-binding protein has an anti-CD 3 sdAb, an anti-ALB sdAb, and an sdAb for DLL 3. This reduces the size of the exemplary DLL3 trispecific antigen-binding protein to below 60 kDa. Thus, in some embodiments, the domains of DLL3 targeted trispecific proteins are all single domain antibody (sdAb) fragments. It is contemplated that in some embodiments the DLL3 binding protein is relatively small and in some embodiments no more than 25kDa, no more than 20kDa, no more than 15kDa, or no more than 10kDa. In some cases, if the DLL3 binding protein is a peptide or small molecule entity, it is 5kDa or less.

In other embodiments, the DLL3 targeted trispecific proteins described herein comprise Small Molecule Entity (SME) conjugates directed against ALB, DLL3, CD3 or all. SME conjugates are small molecules of an average size of about 500to 2000Da and are linked to DLL 3-targeted trispecific proteins by known methods such as sortase conjugation or conjugation. In these cases, one of the domains of the DLL3 trispecific antigen-binding protein is the sortase recognition sequence LPETG (SEQ ID NO: 1896). To attach the SME conjugate to the DLL 3-trispecific antigen-binding protein with a sortase recognition sequence, the protein is incubated with sortase and the SME conjugate, whereby the sortase attaches the SME conjugate to the recognition sequence. In other embodiments, the domain of DLL 3-targeting trispecific proteins described herein that binds to DLL3 comprises a cysteine knot (knottin) peptide for binding to DLL 3. Cysteine knot is a disulfide stabilized peptide with a cysteine knot scaffold and has an average size of about 3.5kDa. It has been envisaged to bind the cysteine knot to certain tumour molecules such as DLL 3. In further embodiments, the third domain of the DLL 3-targeted trispecific proteins described herein that binds to DLL3 comprises a natural DLL3 ligand.

Another feature of the DLL3 targeted trispecific proteins described herein is that they have a single polypeptide design with their domains having flexible linkages. This eases the generation and manufacture of DLL3 targeted trispecific proteins, as they can be encoded by a single cDNA molecule for easy incorporation into a vector. Furthermore, because the DLL3 targeted trispecific proteins described herein are monomeric single polypeptide chains, there is no chain pairing problem or requirement for dimerization. It is contemplated that the DLL 3-targeted trispecific proteins described herein have a reduced propensity to aggregate, unlike other reported molecules such as bispecific proteins with Fc-gamma immunoglobulin domains.

In the DLL 3-targeted trispecific proteins described herein, in some embodiments, the domains are linked by internal linkers L1 and L2, wherein L1 links the first and second domains of the DLL 3-targeted trispecific protein and L2 links the second and third domains of the DLL 3-targeted trispecific protein. The linkers L1 and L2 have optimized lengths and/or amino acid compositions. 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 linker L1 and/or L2 is "short", i.e., consists of 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in some cases, the internal linker consists of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the internal linker L1 and/or L2 is "long", i.e., consists of 15, 20, or 25 amino acid residues. In some embodiments, these internal 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 internal linkers L1 and L2, peptides were selected that have properties that confer flexibility to DLL 3-targeted trispecific proteins, do not interfere with the binding domain, and are resistant to protease cleavage. For example, glycine and serine residues generally provide protease resistance. Examples of internal linkers suitable for linking DLL3 targeted trispecific proteins 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 IDNo.1814)、(GGGGG)_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, internal linker L1 and/or L2 is (GGGGS) ₄ (SEQ ID No. 1817) or (GGGGS) ₃ (SEQ ID No. 1818). In another embodiment, internal linker L1 and/or L2 is GGGGSGGGS (SEQ ID No. 1808).

In some cases, DLL3 targets domains within a trispecific protein for conjugation using an enzymatic site-specific conjugation method involving the use of mammalian or bacterial transglutaminase. Microbial transglutaminase (mTG) is a versatile tool in modern research and biotechnology. The availability, ease of use, and lack of regulation of calcium and guanosine 5' -triphosphate (GTP) of a large number of relatively pure enzymes make mTG the primary cross-linking enzyme used in the food industry and biotechnology. Presently, mTG is used in many applications to attach 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.

In some examples, DLL 3-targeted trispecific proteins are provided in which one of the domains comprises the receptor glutamine in the constant region, which can then be conjugated to the other domain via a lysine-based linker (e.g., any primary amine that is a substrate for TG enzyme (TGase), comprising alkylamine, oxoamine), wherein conjugation occurs only on one or more receptor glutamine residues present in the targeting moiety outside the antigen binding site (e.g., outside the variable region, in the constant region). Thus, conjugation does not occur on glutamine (at least a portion of the surface exposed glutamine) within the variable region. In some examples, the trispecific protein is formed by reacting one of the domains with a lysine-based linker in the presence of a TG enzyme.

In some embodiments, where DLL3 targets one or more domains within a trispecific binding protein for direct ligation, hybrid vectors are prepared in which the DNA encoding the directly ligated domains are themselves directly linked to each other. In some embodiments, where a linker is used, a hybrid vector is prepared in which DNA encoding a first of the three domains is linked to DNA encoding one end of the first linker moiety and DNA encoding a second of the three domains is linked to the other end of the first linker moiety; in addition, DNA encoding a second domain of the three domains is linked to one end of the second linker moiety and DNA encoding a third domain of the three domains is linked to the other end of the second linker moiety, wherein the first domain, the second domain and the third domain are different, and wherein the first domain, the second domain and the third domain are independently selected from domain a, domain B and domain C. Such connections are made, for example, in series or in three-way connections.

CD3 binding domain

The specificity of the T cell response is mediated by recognition of antigens (displayed in the context of the major histocompatibility complex MHC) by TCRs. As part of the TCR body, CD3 is a protein complex comprising one CD3 gamma chain, one CD3 delta chain and two CD3 epsilon chains present on the cell surface. CD3 associates with all of the α and β chains of the TCR and cd3ζ to form the complete TCR. Aggregation of CD3 on T cells (such as by immobilized anti-CD 3 antibodies) results in T cell activation, similar to engagement of T cell receptors, but independent of the specificity typical of their cloning.

In one aspect, the DLL3 targeted trispecific proteins described herein comprise a domain that specifically binds CD 3. In one aspect, the DLL3 targeted trispecific proteins described herein comprise a domain that specifically binds human CD 3. In some embodiments, a DLL3 targeted trispecific protein described herein comprises a domain that specifically binds to cd3γ. In some embodiments, a DLL3 targeted trispecific protein described herein comprises a domain that specifically binds to cd3δ. In some embodiments, a DLL3 targeted trispecific protein described herein comprises a domain that specifically binds CD3 epsilon.

In further embodiments, a DLL3 targeted trispecific protein described herein comprises a domain that specifically binds to a TCR. In certain instances, the DLL3 targeted trispecific proteins described herein comprise a domain that specifically binds to the alpha chain of a TCR. In certain instances, the DLL3 targeted trispecific proteins described herein comprise a domain that specifically binds to the β chain of a TCR.

In certain embodiments, the DLL 3-targeting trispecific protein CD3 binding domains described herein not only exhibit potent CD3 binding affinity with human CD3, but also exhibit excellent cross-reactivity with the corresponding cynomolgus monkey CD3 protein.

In some embodiments, the CD3 binding domain of the DLL3 trispecific antigen-binding protein may be any domain that binds CD3, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. In some cases it may be advantageous for the CD3 binding domain to be derived from the same species as the end use DLL3 trispecific antigen binding protein. For example, it may be beneficial for the CD3 binding domain of a DLL3 trispecific antigen-binding protein to comprise human or humanized residues from the antigen-binding domain of an antibody or antibody fragment for use in humans.

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-CD 3 binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR 1), light chain complementarity determining region 2 (LC CDR 2), and light chain complementarity determining region 3 (LC CDR 3) of the humanized or human anti-CD 3 binding domain described herein, and/or one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR 1), heavy chain complementarity determining region 2 (HC CDR 2), and heavy chain complementarity determining region 3 (HC CDR 3) of the humanized or human anti-CD 3 binding domain comprises one or more (all three) LC CDRs and one or more (all three) HC CDRs.

In some embodiments, the humanized or human anti-CD 3 binding domain comprises a humanized or human light chain variable region specific for CD3, wherein 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 cases, the light chain framework region is a lambda light chain framework. In other cases, the light chain framework region is a kappa light chain framework.

In some embodiments, the humanized or human anti-CD 3 binding domain comprises a humanized or human heavy chain variable region specific for CD3, wherein the heavy chain variable region specific for CD3 comprises human or non-human heavy chain CDRs in a human heavy chain framework region.

In certain instances, the complementarity determining regions of the heavy and/or light chains are derived from known anti-CD 3 antibodies, such as, for example, moluzumab (muromonab) -CD3 (OKT 3), oxybutyumab (oteliximab) (TRX 4), teplizumab (MGA 031), velocimab (visilizumab) (Nuvion), SP34, TR-66 or X35-3、VIT3、BMA030(BW264/56)、CLB-T3/3、CRIS7、YTH12.5、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.

In one embodiment, the anti-CD 3 binding domain is a single chain variable fragment (scFv) comprising the light and heavy chains of the amino acid sequences provided herein. As used herein, "single chain variable fragment" or "scFv" refers to an antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are linked consecutively via a short flexible polypeptide linker and are capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. In one embodiment, the anti-CD 3 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 no more than 30, 20, or 10 modifications (e.g., substitutions) to the amino acid sequences of the light chain variable regions provided herein, or a sequence having 95-99% identity to the amino acid sequences 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 no more than 30, 20, or 10 modifications (e.g., substitutions) to the amino acid sequences of the heavy chain variable regions provided herein, or a sequence having 95-99% identity to the amino acid sequences provided herein. In some examples, the anti-CD 3 binding domain comprises a sequence selected from SEQ ID No.1793-SEQ ID No.1807, or a sequence having 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% identity to a sequence selected from SEQ ID No. 1793-1807. In some examples, the anti-CD 3 binding domain comprises three heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR 3) and three light chain CDRs. The heavy chain CDR1 (HC CDR 1) of the CD3 binding domain comprises a sequence selected from SEQ ID nos. 1820-1831 or comprises one or more modified or substituted sequences in the sequence selected from SEQ ID nos. 1820-SEQ ID nos. 1831, or at least about 80% to about 99%. The heavy chain CDR2 (HC CDR 2) of the CD3 binding domain comprises a sequence selected from SEQ ID No.1832-SEQ ID No.1841 or comprises one or more modified or substituted sequences in the sequence selected from SEQ ID No.1832-SEQ ID No. 1841. The heavy chain CDR3 (HC CDR 3) of the CD3 binding domain comprises a sequence selected from SEQ ID No.1842-SEQ ID No.1853 or comprises one or more modified or substituted sequences in the sequence selected from SEQ ID No.1842-SEQ ID No. 1853. The light chain CDR1 (LC CDR 1) of the CD3 binding domain comprises a sequence selected from SEQ ID No.1852-SEQ ID No.1864 or comprises one or more modified or substituted sequences in the sequence selected from SEQ ID No.1852-SEQ ID No. 1864. The light chain CDR2 (LC CDR 2) of the CD3 binding domain comprises a sequence selected from SEQ ID No.1865-SEQ ID No.1877 or comprises one or more modified or substituted sequences in the sequence selected from SEQ ID No.1865-SEQ ID No. 1877. The light chain CDR3 (LC CDR 3) of the CD3 binding domain comprises a sequence selected from SEQ ID No.1878-SEQ ID No.1884 or comprises one or more modified or substituted sequences in the sequence selected from SEQ ID No.1878-SEQ ID No. 1884. In one embodiment, the humanized or human anti-CD 3 binding domain is an scFv and the light chain variable region comprising an amino acid sequence described herein is attached to the heavy chain variable region comprising an amino acid sequence described herein via an scFv linker. The light chain variable region and the heavy chain variable region of the scFv may 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.

In some cases, the scFv that binds CD3 is prepared according to known methods. For example, scFv molecules may be produced by joining VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise scFv linkers (e.g., ser-Gly linkers) with optimized length and/or amino acid composition. Thus, in some embodiments, the length of the scFv linker is such that the VH or VL domain can intermolecularly associate with other variable domains to form a CD3 binding site. In certain embodiments, such scFv linkers are "short", i.e., consist of 0, 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in some cases, an scFv linker consists of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the scFv linker is a peptide bond. In some embodiments, the 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 scFv linkers, peptides are chosen that confer flexibility, do not interfere with the variable domains and allow interchain folding to bring the two variable domains together to form a functional CD3 binding site. For example, scFv linkers comprising glycine and serine residues generally provide protease resistance. In some embodiments, the linker in the scFv comprises glycine and serine residues. For example, the amino acid sequence of the scFv linker can be optimized by phage display methods to increase CD3 binding and yield of scFv. Examples of peptide scFv linkers suitable for linking the variable light and variable heavy domains in a scFv include, but are not limited to (GS)_n(SEQ IDNo.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)、(GGGGG)_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 may be (GGGGS) ₄ (SEQ ID No. 1817) or (GGGGS) ₃ (SEQ ID No. 1818). In some embodiments, the linker comprises a sequence consisting of any combination of linkers as set forth in SEQ ID No.1809 to SEQ ID No.1818, and in some examples, such linkers are up to 15 amino acids or longer than 15 amino acids in length. Variations in linker length can retain or enhance activity, resulting in superior efficacy in activity studies.

In some embodiments, the CD3 binding domain of the DLL3 targeting trispecific antigen-binding protein has an affinity for CD3 on a CD3 expressing cell, wherein K _D is 1000nM or less, 500nM or less, 200nM or less, 100nM or less, 80nM or less, 50nM or less, 20nM or less, 10nM or less, 5nM or less, 1nM or less, or 0.5nM or less. In some embodiments, the CD3 binding domain of the DLL3 targeting trispecific antigen-binding protein has an affinity for CD3 epsilon, gamma, or delta, wherein K _D is 1000nM or less, 500nM or less, 200nM or less, 100nM or less, 80nM or less, 50nM or less, 20nM or less, 10nM or less, 5nM or less, 1nM or less, or 0.5nM or less. In further embodiments, the CD3 binding domain of the DLL 3-targeting trispecific antigen-binding protein has a low affinity for CD3, i.e., about 100nM or greater.

The affinity for binding to CD3 can be determined, for example, by the ability of DLL3 to target CD3 binding of the trispecific antigen-binding protein itself or its CD3 binding domain to a CD3 binding protein coated on an assay plate, displayed on the surface of a microbial cell, in solution, etc. The binding activity of the DLL 3-targeted trispecific antigen-binding proteins of the present disclosure, per se, or CD3 binding domains thereof, to CD3 can be determined by immobilizing a ligand (e.g., CD 3) or DLL 3-targeted trispecific antigen-binding protein, per se, or CD3 binding domains thereof, to a bead, matrix, cell, or the like. The agent may be added to an appropriate buffer and the binding partner incubated at a given temperature for a period of time. After washing to remove unbound material, the bound protein may be released, e.g., with SDS, a buffer with a high pH, etc., and analyzed, e.g., by Surface Plasmon Resonance (SPR).

Half-life extending domains

Contemplated herein are domains that extend the half-life of an antigen binding domain. Such domains are contemplated to include, but are not limited to, albumin binding domains, fc domains, small molecules, and other half-life extending domains known in the art.

Human Albumin (ALB) (molecular mass 67 kDa) is the most abundant protein in plasma, exists at about 50mg/ml (600. Mu.M), and has a half-life of about 20 days in humans. ALB is used to maintain plasma pH, aids in colloidal blood pressure, acts as a carrier for many metabolites and fatty acids, and serves as the primary drug transporter in plasma.

Non-covalent association with albumin increases the elimination half-life of short-lived proteins. For example, recombinant fusion of albumin binding domain to Fab fragment results in 25-and 58-fold in vivo clearance and 26-and 37-fold half-life extension when administered intravenously to mice and rabbits, respectively, as compared to Fab fragment administration alone. In another example, a sustained effect is observed when insulin is acylated with fatty acids to promote association with albumin when subcutaneously injected in rabbits or pigs. Taken together, these studies demonstrate a link between albumin binding and prolongation.

In one aspect, the DLL3 targeted trispecific proteins described herein comprise a half-life extending domain, such as a domain that specifically binds ALB. In some embodiments, the ALB binding domain that targets DLL3 to a trispecific antigen-binding protein may be any domain that binds ALB, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. In some embodiments, the ALB binding domain is a single chain variable fragment (scFv), single domain antibody (such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain of a camelid-derived single domain antibody (VHH)), a peptide, a ligand, or a small molecule entity that is specific for HSA. In certain embodiments, the ALB binding domain is a single domain antibody. In other embodiments, the HSA binding domain is a peptide. In further 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 relatively small and in some embodiments, no more than 25kD, no more than 20kDa, no more than 15kDa, or no more than 10kDa. In some cases, if the ALB binding domain is a peptide or small molecule entity, it is 5kDa or less.

The half-life extending domain of the DLL3 targeted trispecific antigen binding protein provides altered pharmacodynamics and pharmacokinetics of the DLL3 targeted trispecific antigen binding protein itself. As described above, the half-life extending domain extends the elimination half-life. The half-life extending domain also alters pharmacodynamic properties including altering tissue distribution, penetration and diffusion of the trispecific antigen-binding protein. In some embodiments, the half-life extending domain provides improved tissue (including tumor) targeting, tissue distribution, tissue penetration, tissue in-diffusion, and enhanced efficacy compared to a protein without the half-life extending domain. In one embodiment, the method of treatment effectively and efficiently utilizes a reduced amount of trispecific antigen-binding protein, thereby producing reduced side effects, such as reduced cytotoxicity of non-tumor cells.

Furthermore, the binding affinity of the half-life extending domain may be selected to target a specific elimination half-life in a specific trispecific antigen-binding protein. Thus, in some embodiments, the half-life extending domain has a high binding affinity. In other embodiments, the half-life extending domain has a moderate binding affinity. In other embodiments, the half-life extending domain has low or marginal binding affinity. Exemplary binding affinities include KD concentrations of 10nM or less (high), between 10nM and 100nM (medium), and greater than 100nM (low). As described above, the binding affinity for ALB is determined by known methods such as Surface Plasmon Resonance (SPR). In some embodiments, the ALB binding domains described herein comprise single domain antibodies.

In some embodiments, the half-life extending domain comprises a sequence selected from SEQ ID No.1769-SEQ ID No.1778, or a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to a sequence selected from SEQ ID No.1769-SEQ ID No. 1778. In some examples, the half-life extending domain comprises three heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR 3) and three light chain CDRs. In some examples, the half-life extending domain comprises three heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR 3) or three light chain CDRs. In some embodiments, the heavy chain CDR1 (HC CDR 1) of the half-life extending domain comprises a sequence selected from SEQ ID No.1782-SEQ ID No.1784, or comprises one or more modified or substituted sequences in the sequence selected from SEQ ID No.1782-SEQ ID No.1784, or at least about 80% to about 99%. In some embodiments, the heavy chain CDR2 (HC CDR 2) of the half-life extending domain comprises a sequence selected from SEQ ID No.1785-SEQ ID No.1790, or comprises one or more modified or substituted sequences in the sequence selected from SEQ ID No.1785-SEQ ID No. 1790. The heavy chain CDR3 (HC CDR 3) of the CD3 binding domain comprises a sequence selected from SEQ ID No.1791 or SEQ ID No.1792 or comprises one or more modified or substituted sequences in the sequence selected from SEQ ID No.1791 or SEQ ID No. 1792.

DLL3 binding domains

DLL3 (also known as delta-like ligand 3 or SCDO 1) is a member of the delta-like family of Notch DSL ligands. Representative DLL3 protein orthologs include, but are not limited to, humans (accession numbers np_058637 and np_ 982353), chimpanzees (accession number xp_ 003316395), mice (accession number np_ 031892), and rats (accession number np_ 446118). In humans, the DLL3 gene consists of a stretch of 9.5kbp8 exons located on chromosome 19q 13. Alternate splicing within the last exon resulted in two processed transcripts, one 2389 bases (accession No. nm_ 016941) and one 2052 bases (accession No. nm_ 203486). The former transcript encodes a 618 amino acid protein (accession No. np_058637) and the latter transcript encodes a 587 amino acid protein (accession No. np_ 982353). These two protein isoforms of DLL3 have 100% identity overall in their extracellular domains and in their transmembrane domains, the only difference being that the longer isoform contains an extended cytoplasmic tail containing 32 additional residues at the carboxy terminus of the protein. The extracellular region of DLL3 protein comprises six EGF-like domains, a single DSL domain and an N-terminal domain. In general, EGF domains are thought to be present at about 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) of hDLL3, DSL domains are present at about amino acid residues 176-215, and N-terminal domains are present at about amino acid residues 27-175. Each of the EGF-like domain, DSL domain and N-terminal domain comprises a portion of a DLL3 protein as defined by different amino acid sequences. In some embodiments, the EGF-like domain is referred to as EGF1 to EGF6, with EGF1 being proximal to the N-terminal portion of the protein. Generally, DSL ligands consist of a series of structural domains: the unique N-terminal domain is followed by a conserved DSL domain, a plurality of tandem Epidermal Growth Factor (EGF) -like repeats, a transmembrane domain, and a cytoplasmic domain that is not highly conserved within the ligand but contains a plurality of lysine residues, which are potential sites for ubiquitination of the unique E3 ubiquitin ligase. DSL domains are degenerate EGF domains that are necessary but not sufficient for interaction with Notch receptors. Furthermore, the first two EGF-like repeats of most DSL ligands contain a smaller protein sequence motif, termed DOS domain, which interacts in a cooperative manner with the DSL domain when Notch signaling is activated.

In some embodiments, the DLL3 trispecific binding proteins disclosed in the present disclosure are generated, manufactured, engineered, or selected to react with a selected domain, motif, or epitope within the DLL3 protein. In some embodiments, DLL3 targets a trispecific protein binding DSL domain, and in some embodiments it binds an epitope within the DSL domain comprising G203, R205, P206.

In some embodiments, the DLL3 binding domain of the DLL3 targeting trispecific proteins of the present disclosure is engineered, manufactured, and/or selected to react with both isoforms of DLL3 or a single isoform of a protein, or conversely, comprises a pan DLL binding domain that reacts or associates with at least one additional DLL family member other than DLL 3. In some embodiments, DLL3 binding domains (e.g., DLL3 binding domains) are engineered, manufactured, and/or selected such that they react with domains (or epitopes thereof) exhibited solely by DLL3 or with domains that are at least somewhat conserved within multiple or all DLL family members.

In some embodiments, the DLL3 binding domain associates or binds to a particular epitope, portion, motif or domain of DLL 3. Both DLL3 isoforms incorporate the same extracellular region comprising at least an N-terminal domain, a DSL (delta/Serrate/lag-2) domain and six EGF-like domains (i.e., EGF1-EGF 6). Thus, in certain embodiments, the DLL3 binding domain binds to or associates with the N-terminal domain of DLL3 (amino acids 27-175 in 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 disclosure, the DLL3 binding domain associates or binds with a particular epitope located in a particular EGF-like domain of DLL 3. In some embodiments, the DLL3 binding domain associates or binds with 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 foregoing domains comprises more than one epitope and/or more than one bin (bin).

In some embodiments, the DLL3 binding domain binds, reacts with, or associates with the DSL domain or an epitope therein. In other embodiments, the DLL3 binding domain binds, reacts with, or associates with a particular EGF-like domain or epitope therein. In some embodiments, the DLL3 binding domain binds, reacts with, or associates with the N-terminal domain or an epitope therein.

In some embodiments, a DLL3 binding protein of the present disclosure (e.g., a DLL3 binding domain of a trispecific protein of the present disclosure) binds to a full-length DLL3 protein or a fragment thereof (e.g., an epitope-containing fragment within a full-length DLL3 protein), as described above. In some cases, the epitope-containing fragments comprise antigenic or immunogenic fragments of DLL3 proteins and derivatives thereof. In some embodiments, the epitope-containing fragment (including antigenic or immunogenic fragments) 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 length of the whole protein, 90% or more, 75%, or 50%, or 25%, or 10% or more of the length of the whole protein. In some embodiments, epitope-containing fragments (including antigenic or immunogenic fragments) of DLL3 are capable of eliciting a related immune response in a patient. In some embodiments, derivatives of DLL3 include variants in the sequence in which one or more (e.g., 1-20, e.g., 15 amino acids, or up to 20%, e.g., up to 10% or 5% or 1%) deletions, insertions, or substitutions are made to the DLL3 sequence provided in SEQ ID No.1885 (UniProtKB accession No. Q9NYJ 7), by amino acid number based on the total length of the protein. In some embodiments, the substitutions comprise conservative substitutions. In some examples, derivatives and variants of DLL3 have substantially the same biological function as the DLL3 protein from which they are derived. For example, in some cases, derivatives and variants of DLL3 have comparable antigenicity or immunogenicity to the proteins they are derived from, have ligand binding activity of the proteins they are derived from, or active receptor complex forming ability, or preferably both, and have the same tissue distribution as DLL 3.

The design of the DLL3 targeted trispecific proteins described herein makes the binding domain to DLL3 flexible, as the binding domain to DLL3 can be any type of binding domain, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. In some embodiments, the binding domain to 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 of a camelid derived single domain antibody (VHH). In other embodiments, the binding domain for DLL3 is a non-Ig binding domain, i.e., an antibody mimetic, such as ANTICALIN, AFFILIN, an affibody molecule, affimer, affitin, alphabody, avimer, DARPin, fynomer, a kunitz domain peptide, and a monoclonal antibody. In further embodiments, the binding domain for DLL3 is a ligand or peptide that binds or associates with DLL 3. In further embodiments, the binding domain for DLL3 is a cysteine knot. In further embodiments, the binding domain for DLL3 is a small molecule entity.

In some embodiments, the DLL3 binding domain binds a protein comprising the sequence SEQ ID No.1885 (UniProtKB accession No. Q9NYJ 7). In some embodiments, the DLL3 binding domain binds a protein comprising a truncated sequence compared to SEQ ID No.1885 (UniProtKB accession No. Q9NYJ 7). In some embodiments, the DLL3 binding domain binds a protein comprising the sequence SEQ ID No.1892 or SEQ ID No.1893 (which is the mature extracellular domain of a 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.

In some embodiments, the DLL3 binding domain is an anti-DLL 3 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-DLL 3 antibodies described herein are contemplated. For example, in certain embodiments, it is contemplated that amino acid sequence variants of the anti-DLL 3 antibodies described herein may increase the binding affinity and/or other biological properties of the antibodies. Exemplary methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody.

Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics (antigen binding). In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the 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 screened to obtain the desired activity, retention/improved antigen binding, reduced immunogenicity or improved T cell mediated cytotoxicity (TDCC). To prepare antibody variants, conservative and non-conservative amino acid substitutions are contemplated.

In another example of a substitution to produce a variant anti-DLL 3 antibody, one or more hypervariable region residues of the parent antibody are substituted. In general, variants are then selected based on improvements in desired properties compared to the parent antibody, e.g., increased affinity, reduced immunogenicity, increased binding pH dependence.

In some embodiments, the DLL3 binding domain of the DLL3 targeting trispecific protein is a single domain antibody, such as a heavy chain variable domain (VH) specific for DLL3, 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 DLL 3-targeted trispecific proteins described herein is any domain that binds DLL3, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. 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.

In general, it should be noted that the term single domain antibody as used herein is not limited in its broadest sense to a particular biological source or a particular method of preparation. 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 do not naturally contain light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. The single domain antibody may be any single domain antibody in the prior art, or any future single domain antibody. The single domain antibody may be derived from any species including, but not limited to, mouse, human, camel, llama, goat, rabbit, cow. For example, in some embodiments, a single domain antibody of the present disclosure is obtained by: (1) By isolating the VHH domain of a naturally occurring heavy chain antibody; (2) By expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) By "humanizing" naturally occurring VHH domains or by expressing nucleic acids encoding such humanized VHH domains; (4) By "camelizing" naturally occurring VH domains from any animal species, particularly from mammalian species, such as from humans, or by expressing nucleic acids encoding such camelized VH domains; (5) By "camelizing", "domain antibodies" or "dabs", or by expressing nucleic acids encoding such camelized VH domains; (6) Preparing a protein, polypeptide or other amino acid sequence by using synthetic or semi-synthetic techniques; (7) Preparing a nucleic acid encoding a single domain antibody by using nucleic acid synthesis techniques known in the art, and then expressing the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing.

In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody directed against DLL 3. As further described herein, such VHH sequences can generally be generated or obtained by: suitably immunizing a llama species with DLL3 (i.e., to generate an immune response and/or heavy chain antibodies against DLL 3); obtaining a suitable biological sample (such as a blood sample, serum sample or B-cell sample) from the llama; and generating VHH sequences for DLL3 starting from the sample using any suitable technique known in the art.

In another embodiment, such naturally occurring VHH domain directed against DLL3 is derived from a natural library of camelid VHH sequencesLibrary), such as by screening such libraries using DLL3 or at least one portion, fragment, epitope or epitope thereof, using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semisynthetic libraries derived from natural VHH libraries are used, such as VHH libraries obtained from natural VHH libraries by techniques such as random mutagenesis and/or CDR shuffling as described for example in WO 00/43507.

In another embodiment, yet another technique for obtaining VHH sequences for DLL3 involves suitably immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., to generate an immune response and/or heavy chain antibodies for DLL 3), obtaining a suitable biological sample (such as a blood sample, serum sample, or B-cell sample) from the transgenic mammal, and then starting from the sample using any suitable technique known in the art, generating VHH sequences for DLL 3. For example, rats or mice expressing heavy chain antibodies as described in WO 02/085945 and WO 04/049794, as well as further methods and techniques, may be used for this purpose.

In some embodiments, the DLL3 targeted anti-DLL 3 single domain antibody of the trispecific protein comprises a single domain antibody whose amino acid sequence corresponds to that of a naturally occurring VHH domain, but has been "humanized", i.e., by replacement of one or more amino acid residues in the naturally occurring VHH sequence (particularly the framework sequence) with one or more amino acid residues (e.g., as shown above) present at corresponding positions in the VH domain of a conventional 4-chain antibody from a human. This can be done in a manner known in the art, which will be clear to the skilled person, for example based on the further description herein. Also, it should be noted that such humanized anti-DLL 3 single domain antibodies of the present disclosure are obtained in any suitable manner known per se (i.e., as indicated at points (1) - (8) above), and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VHH domains as starting materials. In some further embodiments, an anti-DLL 3 single domain antibody as described herein comprises a single domain antibody having an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but has been "camelized", i.e., by replacement of one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody with one or more amino acid residues present at corresponding positions in the VHH domain of the heavy chain antibody. Such "camelized" substitutions are preferably inserted at amino acid positions formed and/or present at the VH-VL interface and/or at so-called camelidae marker residues (see e.g. WO 94/04678 and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence used as a starting material or origin for the production or design of the camelised single domain is preferably a VH sequence from a mammal, more preferably a human, such as a VH3 sequence. However, it should be noted that in certain embodiments, such camelized anti-DLL 3 single domain antibodies of the present disclosure are obtained in any suitable manner known in the art (i.e., as indicated at points (1) - (8) above), and are therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VH domain as starting material. For example, as further described herein, "humanization" and "camelization" are both performed by the following operations: providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then altering one or more codons in the nucleotide sequence in a manner such that the new nucleotide sequence encodes a "humanized" or "camelized" single domain antibody, respectively. This nucleic acid can then be expressed, thereby providing the desired anti-DLL 3 single domain antibodies of the present disclosure. Alternatively, in other embodiments, the amino acid sequences of the desired humanized or camelized anti-DLL 3 single domain antibodies of the present disclosure are designed based on the amino acid sequences of naturally occurring VHH domains or VH domains, respectively, and then synthesized de novo using known peptide synthesis techniques. In some embodiments, the nucleotide sequences encoding the desired humanized or camelized anti-DLL 3 single domain antibodies of the present disclosure are designed based on the amino acid sequences or nucleotide sequences of naturally occurring VHH domains or VH domains, respectively, and then synthesized de novo using known nucleic acid synthesis techniques, followed by expression of the nucleic acids thus obtained using known expression techniques, thereby providing the desired anti-DLL 3 single domain antibodies of the present disclosure.

Other suitable methods and techniques for obtaining an anti-DLL 3 single domain antibody of the present disclosure and/or a nucleic acid encoding the same, starting from a naturally occurring VH sequence or VHH sequence, include, for example, combining one or more portions 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 portions 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 semisynthetic sequences in a suitable manner so as to provide an anti-DLL 3 single domain antibody of the present disclosure or a nucleotide sequence or nucleic acid encoding the same.

In some embodiments, the DLL3 binding domain is an anti-DLL 3 specific antibody comprising a heavy chain variable complementarity determining region CDR1, a heavy chain variable CDR2, a heavy chain variable CDR3, a light chain variable CDR1, a light chain variable CDR2, and a light chain variable CDR3. In some embodiments, the DLL3 binding domain comprises any domain that binds DLL3, including, but not limited to, domains from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies or antigen-binding fragments such as single domain antibodies (sdabs), fab', F (ab) 2, and Fv fragments, fragments composed of one or more CDRs, single chain antibodies (e.g., single chain Fv fragments (scFv)), disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers composed 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-DLL 3 single domain antibody comprises heavy chain variable Complementarity Determining Regions (CDRs) CDR1, CDR2, and CDR3.

In some embodiments, the DLL3 binding domain is a polypeptide comprising an amino acid sequence comprising four framework regions/sequences (f 1-f 4) separated by three complementarity determining regions/sequences, as shown in the following formula: f1-r1-f2-r2-f3-r3-f4, wherein 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 present disclosure comprise, 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 region comprises, 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 comprises an amino acid sequence selected from SEQ ID No.1-SEQ ID No.442 and SEQ ID No. 1886. In some embodiments, CDR1 of the DLL3 binding domain comprises a sequence selected from SEQ ID No.443-SEQ ID No.884 and SEQ ID No.1887, or one or more amino acid substitutions relative to a sequence selected from SEQ ID No.443-SEQ ID No.884 and SEQ ID No. 1887. In some embodiments, CDR2 comprises a sequence selected from SEQ ID No.885-SEQ ID No.1326 and SEQ ID No.1888, or one or more amino acid substitutions relative to a sequence selected from SEQ ID No.885-SEQ ID No.1326 and SEQ ID No. 1888. In some embodiments, CDR3 comprises a sequence selected from SEQ ID No.1327-SEQ ID No.1768 and SEQ ID No.1889, or one or more substitutions relative to a sequence selected from SEQ ID No.1327-SEQ ID No.1768 and SEQ ID No. 1889.

In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID No.443-SEQ ID No.884 and SEQ ID No.1887, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in an amino acid sequence selected from SEQ ID No.443-SEQ ID No.884 and SEQ ID No. 1887. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID No.885-SEQ ID No.1326 and SEQ ID No.1888, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in an amino acid sequence selected from SEQ ID No.885-SEQ ID No.1326 and SEQ ID No. 1888. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID No.1327-SEQ ID No.1768 and SEQ ID No.1889, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in a sequence selected from SEQ ID No.1327-SEQ ID No.1768 and SEQ ID No. 1889.

In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID No.495-SEQ ID No.528, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid sequence selected from SEQ ID No.495-SEQ ID No. 528. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID No.937-SEQ ID No.970, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid sequence selected from SEQ ID No.937-SEQ ID No. 970. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID No.1379-SEQ ID No.1412, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in a sequence selected from SEQ ID No.1379-SEQ ID No. 1412.

In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID No.529-SEQ ID No.809, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in the amino acid sequence selected from SEQ ID No.529-SEQ ID No. 809. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID No.971 to SEQ ID No.1251, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid sequence selected from SEQ ID No.971 to SEQ ID No. 1251. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID nos. 1379 to 1412, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in a sequence selected from SEQ ID nos. 1379-1412.

In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID No.810-SEQ ID No.884, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid sequence selected from SEQ ID No.810-SEQ ID No. 884. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID No.1252 to SEQ ID No.1326, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid sequence selected from SEQ ID No.1252 to SEQ ID No. 1326. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID No.1692 to SEQ ID No.1768, or a variant having one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in a sequence selected from SEQ ID No.1692 to SEQ ID No. 1768.

In various embodiments, the DLL3 binding domains of the present disclosure have 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% identity 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 has 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% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:53-SEQ ID No. 86.

In various embodiments, the DLL3 binding domain of the present disclosure has 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% identity to an amino acid sequence selected from SEQ ID No.87-SEQ ID No. 367.

In various embodiments, the DLL3 binding domain of the present disclosure has 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% identity to SEQ ID No.68 or a sequence derived from SEQ ID No. 68.

In various embodiments, the DLL3 binding domain of the present disclosure has 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% identity to SEQ ID No.75 or a sequence derived from SEQ ID No. 75.

In some embodiments, DLL3 targets the DLL3 binding domain of the trispecific binding protein to cross-react with human and cynomolgus monkey DLL3. In some embodiments, the DLL3 binding domain is specific for human DLL3. In certain embodiments, the DLL3 binding domains disclosed herein bind human DLL3 with human Kd (hKd). In certain embodiments, the DLL3 binding domains disclosed herein bind cynomolgus monkey DLL3 with cynomolgus monkey Kd (cKd). In certain embodiments, the DLL3 binding domains disclosed herein bind cynomolgus monkey DLL3 and human DLL3 with cynomolgus monkey Kd (cKd) and human Kd (hKd), respectively. In some embodiments, the DLL3 binding protein binds to human and cynomolgus monkey DLL3 with comparable binding affinities (i.e., hKd and cKd values differ by no more than ±10%). In some embodiments, hKd and cKd range from about 0.001nM to about 500nM. In some embodiments, hKd and cKd range from about 0.001nM to about 450nM. In some embodiments, hKd and cKd range from about 0.001nM to about 400nM. In some embodiments, hKd and cKd range from about 0.001nM to about 350nM. In some embodiments, hKd and cKd range from about 0.001nM to about 300nM. In some embodiments, hKd and cKd range from about 0.001nM to about 250nM. In some embodiments, hKd and cKd range from about 0.001nM to about 200nM. In some embodiments, hKd and cKd range from about 0.001nM to about 150nM. In some embodiments, hKd and cKd range from about 0.001nM to about 100nM. In some embodiments, hKd and cKd range from about 0.1nM to about 90nM. In some embodiments, hKd and cKd range from about 0.2nM to about 80nM. In some embodiments, hKd and cKd range from about 0.3nM to about 70nM. In some embodiments, hKd and cKd range from about 0.4nM to about 50nM. In some embodiments, hKd and cKd range from about 0.5nM to about 30nM. In some embodiments, hKd and cKd range from about 0.6nM to about 10nM. In some embodiments, hKd and cKd range from about 0.7nM to about 8nM. In some embodiments, hKd and cKd range from about 0.8nM to about 6nM. In some embodiments, hKd and cKd range from about 0.9nM to about 4nM. In some embodiments, hKd and cKd range from about 1nM to about 2nM.

In certain embodiments, the DLL3 binding domain of the present disclosure preferentially binds to 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 in or on the cell membrane surface of the cell in which DLL3 is or has been expressed. In some cases, the soluble DLL3 is present in the subject's blood and/or lymphatic circulation. In one embodiment, the DLL3 binding protein binds to the 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 compared to the soluble DLL3. In one embodiment, the antigen binding proteins of the present disclosure bind membrane-bound DLL3 preferentially 30-fold over soluble DLL3. Determination of preferential binding of antigen binding proteins to membrane-bound DLL3 over soluble DLL3 can be readily determined using assays well known in the art.

In some embodiments, any of the foregoing DLL3 binding domains (e.g., anti-DLL 3 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).

In some embodiments, any of the foregoing DLL3 binding domains (e.g., anti-DLL 3 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). Integration into Chimeric Antigen Receptor (CAR)

In certain examples, the DLL3 targeted trispecific antigen-binding proteins of the present disclosure can be incorporated into a Chimeric Antigen Receptor (CAR). Engineered immune effector cells (T cells or NK cells) can be used to express CARs comprising anti-DLL 3 targeted trispecific proteins containing anti-DLL 3 single domain antibodies as described herein. In one embodiment, a CAR comprising an anti-DLL 3 targeted trispecific protein as described herein is linked via a hinge region to a transmembrane domain and also to a costimulatory domain, a functional signaling domain obtained from OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278) or 4-1 BB. In some embodiments, the CAR further comprises a sequence encoding an intracellular signaling domain, such as 4-1BB and/or CD3 ζ.

Characteristics of reducing tumor growth

In certain embodiments, the DLL 3-targeted trispecific proteins of the present disclosure reduce tumor cell growth in vivo when administered to a subject having DLL 3-expressing tumor cells. The measurement of tumor cell growth reduction can be determined by a number of different methods well known in the art. Non-limiting examples include direct measurement of tumor size, measurement of resected tumor mass and comparison to a control subject, measurement by imaging techniques (e.g., CT or MRI) that may or may not use isotopes or luminescent molecules (e.g., luciferase) for enhanced analysis, and the like.

In particular 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% reduction in vivo growth of tumor cells, as compared to a control antigen binding agent, with a reduction in tumor growth of about 100% indicating complete response and tumor disappearance. In further embodiments, administration of the trispecific protein of the present disclosure results in a reduction in tumor cell growth in vivo of about 50-100%, about 75-100%, or about 90-100% as compared to a control antigen binding agent. In further embodiments, administration of the trispecific protein of the present disclosure results in a reduction in tumor cell growth in vivo of about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100% as compared to a control antigen binding agent. DLL3 targeted trispecific protein modification

The DLL3 targeted trispecific proteins described herein encompass derivatives or analogues in which (i) the amino acid is substituted with an amino acid residue not encoded by the genetic code, (ii) the mature polypeptide is fused to another compound such as polyethylene, (iii) additional amino acids are fused to the protein such as a leader or secretory sequence or a sequence for purification of the protein.

Typical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a 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 such as arginylation and ubiquitination to the targeting protein.

Modifications are made anywhere in the DLL3 targeted trispecific proteins described herein, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Some common peptide modifications that can be used to modify DLL3 targeted trispecific proteins include glycosylation, lipid ligation, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blocking of amino or carboxyl groups in polypeptides or both by covalent modification, and ADP-ribosylation.

In some embodiments, the derivative of a DLL3 targeted trispecific protein as described herein comprises an immunoreactive modulator derivative and an antigen binding molecule comprising one or more modifications.

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 a target molecule or a specific location or locus on the target molecule. When the antibody is monovalent, each binding site of the molecule will specifically bind to a single antigen position or epitope. When an antibody comprises more than one target binding site (multivalent), each target binding site may specifically bind to the same or different molecules (e.g., may bind to different ligands or different antigens, or different epitopes or positions on the same antigen).

In some embodiments, the DLL3 targeted trispecific proteins of the present disclosure contain, inter alia, one or more additional amino acid residue substitutions, mutations and/or modifications that result in compounds with preferred characteristics including, but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, reduced immunogenicity, increased yield, altered Fc ligand binding to Fc receptor (FcR), enhanced or reduced "ADCC" (antibody dependent cell mediated cytotoxicity) or "CDC" (complement dependent cytotoxicity) activity, altered glycosylation and/or disulfide bonds, and modified binding specificity. In some cases, these DLL3 targeted trispecific protein variants are advantageously used to enhance the effective anti-tumor properties of the disclosed DLL3 targeted trispecific proteins.

In some embodiments, the DLL3 targeted trispecific proteins of the present disclosure have a half-life in a mammal (e.g., a human or cynomolgus monkey) of less than about 5 days, greater than 10 days, greater than about 15 days, greater than about 20 days, greater than about 25 days, greater than about 30 days, greater than about 35 days, greater than about 40 days, greater than about 45 days, greater than about 2 months, greater than about 3 months, greater than about 4 months, or greater than about 5 months. In some cases, an increase in half-life results in higher serum titers, thus reducing the frequency of administration of DLL3 targeted trispecific proteins, reducing the concentration of antibodies to be administered, or both.

Other embodiments comprise one or more engineered glycoforms (glycoforms), i.e., DLL 3-targeted trispecific binding proteins comprising altered glycosylation patterns or altered carbohydrate compositions, covalently linked to a protein. In some cases, the engineered glycoforms can be used for a variety of purposes including, but not limited to, enhancing or reducing effector function, increasing affinity of a trispecific protein for a target, or facilitating the production of a trispecific protein. In certain embodiments where reduced effector function is desired, the molecule is engineered to express a non-glycosylated form. Some embodiments include substitutions that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at the sites. Conversely, in some cases, by engineering one or more additional glycosylation sites, the Fc-containing trispecific proteins of the present disclosure are conferred enhanced effector function or improved binding.

In some cases, DLL 3-targeted trispecific proteins are differentially modified during or after production by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, binding to antibody molecules or other cellular ligands, and the like. Any of a variety of chemical modifications are made by techniques including, but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, naBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, and the like.

The present disclosure also encompasses various post-translational modifications, including, for example, N-linked or O-linked carbohydrate chains, processing of the N-or C-terminus, attachment of chemical moieties to amino acid backbones, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of N-terminal methionine residues due to prokaryotic host cell expression. Furthermore, in some cases, DLL3 targeted trispecific binding proteins are modified with a detectable label, such as an enzyme label, fluorescent label, radioisotope label or affinity label, to allow detection and isolation of the modulator. Polynucleotides encoding DLL3 targeted trispecific proteins

In some embodiments, polynucleotide molecules encoding anti-DLL 3 trispecific binding proteins described herein are also provided. In some embodiments, the polynucleotide molecule is provided as a DNA construct. In other embodiments, the polynucleotide molecule is provided as a messenger RNA transcript.

The polynucleotide molecule is constructed by known methods, such as by combining genes encoding three binding domains separated by peptide linkers or in other embodiments directly linked by peptide bonds into a single genetic construct operably linked to a suitable promoter and optionally a suitable transcription terminator, and expressing the gene in bacteria or other suitable expression systems, e.g., CHO cells. In embodiments where the DLL3 binding domain is a small molecule, the polynucleotide contains a gene encoding a CD3 binding domain and a half-life extending domain. In embodiments where the half-life extending domain is a small molecule, the polynucleotide contains a gene encoding a domain that binds CD3 and DLL 3. Any number of suitable transcription and translation elements may be used, including constitutive and inducible promoters, depending on the vector system and host used. The promoters are selected so that they drive expression of the polynucleotide in the respective host cells.

In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents another embodiment. The recombinant vector may be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, viruses (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, lentiviruses, etc.), and cosmids.

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

Thus, in some embodiments, the DLL3 targeted trispecific proteins described herein are produced by introducing a vector encoding the above proteins into a host cell and culturing the host cell under conditions that express the protein domain, which may be isolated, and optionally further purified.

Pharmaceutical composition

In some embodiments, there is also provided a pharmaceutical composition comprising an anti-DLL 3 trispecific binding protein described herein, a vector comprising a polynucleotide encoding a polypeptide of a DLL3 targeted trispecific protein, or a host cell transformed with such a vector, and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredient and that is non-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 solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and can be administered to a subject in appropriate dosages. Preferably, the composition is sterile. These compositions may also contain adjuvants such as preserving, emulsifying and dispersing agents. Prevention of microbial action can be ensured by the inclusion of various antibacterial and antifungal agents. Another embodiment provides one or more of the DLL3 targeted trispecific proteins described above packaged in lyophilized form or in an aqueous medium.

In some embodiments of the pharmaceutical compositions, the DLL3 targeted trispecific proteins described herein are encapsulated in nanoparticles. 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, DLL3 targets the attachment of a trispecific protein to a liposome. In some cases, DLL3 targets the surface of the liposome to which the trispecific protein is conjugated. In some cases, the DLL3 trispecific antigen-binding protein is encapsulated within the outer shell of a liposome. In some cases, the liposome is a cationic liposome.

The DLL3 targeted trispecific proteins described herein are contemplated for use as pharmaceutical agents. Administration is accomplished by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration in different ways. In some embodiments, the route of administration depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. The dose of any one patient depends on many factors including the patient's size, body surface area, age, sex, the particular compound to be administered, the time and route of administration, the type of therapy, general health, and other drugs administered simultaneously. An "effective dose" refers to an amount of an active ingredient that is sufficient to affect the course and severity of the disease, such that such pathology is reduced or alleviated, and can be determined using known methods.

In some embodiments, DLL3 targeted trispecific proteins of the present disclosure are administered at a dose of up to 10mg/kg at a frequency of once per week. In some cases, the dosage ranges from about 1ng/kg to about 10mg/kg. In some embodiments, the dosage is from about 1ng/kg to about 10ng/kg, from about 5ng/kg to about 15ng/kg, from about 12ng/kg to about 20ng/kg, from about 18ng/kg to about 30ng/kg, from about 25ng/kg to about 50ng/kg, from about 35ng/kg to about 60ng/kg, from about 45ng/kg to about 70ng/kg, from about 65ng/kg to about 85ng/kg, from about 80ng/kg to about 1 μg/kg, from about 0.5 μg/kg to about 5 μg/kg, from about 2 μg/kg to about 10 μg/kg, from about 7 μg/kg to about 15 μg/kg, from about 12 μg/kg to about 25 μg/kg, from about 20 μg/kg to about 50 μg/kg, from about 35 μg/kg to about 70 μg/kg, from about 45 μg/kg to about 80 μg/kg, from about 65 μg/kg to about 90 μg/kg, from about 85 μg/kg to about 0.09 mg to about 0.10 mg. In some cases, the dosage is from about 0.1mg/kg to about 0.2mg/kg, from about 0.25mg/kg to about 0.5mg/kg, from about 0.45mg/kg to about 1mg/kg, from about 0.75mg/kg to about 3mg/kg, from about 2.5mg/kg to about 4mg/kg, from about 3.5mg/kg to about 5mg/kg, from about 4.5mg/kg to about 6mg/kg, from about 5.5mg/kg to about 7mg/kg, from about 6.5mg/kg to about 8mg/kg, from about 7.5mg/kg to about 9mg/kg, or from about 8.5mg/kg to about 10mg/kg. In some embodiments, the frequency of administration is about less than daily, every other day, less than once daily, twice weekly, every 7 days, every two weeks, every three weeks, every four weeks, or monthly. In some cases, the frequency of administration is weekly. In some cases, the frequency of administration is weekly, and the dosage is up to 10mg/kg. In some cases, the duration of administration is from about 1 day to about 4 weeks or more.

In some embodiments, DLL3 of the present disclosure targets a dose of trispecific protein at about 1 μg to about 100 μg, about 1 μg to about 500 μg, about 1 μg to about 1mg, about 1 μg to about 2mg, about 1 μg to about 5mg, about 1 μg to about 10mg, about 1 μg to about 100mg, about 100 μg to about 500 μg, about 100 μg to about 1mg, about 100 μg to about 2mg, about 100 μg to about 5mg, about 100 μg to about 10mg, about 100 μg to about 100mg, about 500 μg to about 1mg, about 500 μg to about 2mg, about 500 μg to about 5mg, about 500 μg to about 10mg, about 500 μg to about 100mg, about 1mg to about 2mg, about 1mg to about 5mg, about 1mg to about 10mg, about 1mg to about 100mg, about 2mg to about 5mg, about 2mg to about 10mg, about 2mg to about 2mg, about 100mg to about 5mg, about 5mg or about 10mg to about 10mg. In some embodiments, the DLL3 targeted trispecific proteins of the present disclosure are 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 μg, about 15 μg to about 3600 μg, about 45 μg to about 135 μg, about 45 μg to about 405 μg, 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 405 μg to about 3600 μg. In some embodiments, the first dose is about 5mg. In some embodiments, the dose is about 7mg. In some embodiments, the dose is about 10mg. In some embodiments, the dose is about 12mg. In some embodiments, the dose is about 15mg. In some embodiments, the dose is about 20mg. In some embodiments, the dose is about 30mg. In some embodiments, the dose is about 40mg. In some embodiments, the dose is about 50mg. In some embodiments, the dose is about 70mg. In some embodiments, the dose is about 100mg.

The DLL3 targeted trispecific proteins described herein may be administered using different dosages. In some embodiments, the DLL3 targeted trispecific proteins of the present disclosure are administered according to a schedule comprising the steps of: (i) Administering a first dose of DLL3 targeted trispecific protein, and (ii) administering a second dose of DLL3 targeted trispecific protein, wherein the second dose is higher than the first dose. In some embodiments, the program further comprises step (iii) administering a third dose of DLL3 targeted trispecific protein, wherein the third dose is greater than the second dose. In some embodiments, the program further comprises step (iv) administering a fourth dose of DLL3 targeted trispecific protein, wherein the fourth dose is higher than the third dose. In some embodiments, the program further comprises step (v) administering a fifth dose of DLL3 targeted trispecific protein, wherein the fifth dose is higher than the fourth dose.

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 1mg, from about 1 μg to about 2mg, from about 1 μg to about 5mg, from about 1 μg to about 8mg, from about 1 μg to about 10mg, from about 1 μg to about 50mg, from about 1 μg to about 100mg, from about 100 μg to about 500 μg, from about 100 μg to about 1mg, from about 100 μg to about 2mg, from about 100 μg to about 5mg, from about 100 μg to about 8mg, from about 100 μg to about 10mg, About 100 μg to about 50mg, about 100 μg to about 100mg, about 500 μg to about 1mg, about 500 μg to about 2mg, about 500 μg to about 5mg, about 500 μg to about 8mg, about 500 μg to about 10mg, about 500 μg to about 50mg, about 500 μg to about 100mg, about 1mg to about 2mg, about 1mg to about 5mg, about 1mg to about 8mg, about 1mg to about 10mg, about 1mg to about 50mg, about 1mg to about 100mg, about 2mg to about 5mg, About 2mg to about 8mg, about 2mg to about 10mg, about 2mg to about 50mg, about 2mg to about 100mg, about 5mg to about 8mg, about 5mg to about 10mg, about 5mg to about 50mg, about 5mg to about 100mg, about 8mg to about 10mg, about 8mg to about 50mg, about 8mg to about 100mg, about 10mg to about 50mg, or about 50mg to about 100mg. 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 3mg. In some embodiments, the first dose is about 4mg. In some embodiments, the first dose is about 5mg. In some embodiments, the first dose is about 6mg. In some embodiments, the first dose is about 7mg. In some embodiments, the first dose is about 8mg. In some embodiments, the first dose is about 9mg. In some embodiments, the first dose is about 10mg. In some embodiments, the first dose is about 11mg. In some embodiments, the first dose is about 12mg. In some embodiments, the first dose is about 15mg. In some embodiments, the first dose is about 20mg. In some embodiments, the first dose is about 30mg. In some embodiments, the first dose is about 40mg. In some embodiments, the first dose is about 50mg. In some embodiments, the first dose is about 70mg. In some embodiments, the first dose is about 100mg.

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

In some embodiments, the first dose is administered once per day, twice per day, three times per day, four times per day, five times per day, six times per day, seven times per day, eight times per day, nine times per day, or ten times per 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 other week, once every three weeks, once every four weeks, or once every five weeks.

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

In some embodiments, the second dose is administered for about 1 week to about 5 weeks, about 1 week to about 10 weeks, about 1 week to about 20 weeks, about 1 week to about 50 weeks, about 1 week to about 80 weeks, about 1 week to about 100 weeks, about 5 weeks to about 10 weeks, about 5 weeks to about 20 weeks, about 5 weeks to about 50 weeks, about 5 weeks to about 80 weeks, about 5 weeks to about 100 weeks, about 10 weeks to about 20 weeks, about 10 weeks to about 50 weeks, about 10 weeks to about 80 weeks, about 10 weeks to about 100 weeks, about 20 weeks to about 50 weeks, about 20 weeks to about 80 weeks, about 20 weeks to about 100 weeks, about 50 weeks to about 80 weeks, about 80 weeks to about 100 weeks, about 1 week to about 9 weeks, about 1 week to about 18 weeks, about 1 week to about 27 weeks, about 1 week to about 36 weeks, about 9 weeks to about 18 weeks, about 9 weeks to about 27 weeks, about 9 weeks to about 36 weeks, about 36 weeks to about 27 weeks, about 36 weeks, or about 36 to about 36 weeks.

In some embodiments, the second dose is administered once per day, twice per day, three times per day, four times per day, five times per day, six times per day, seven times per day, eight times per day, nine times per day, or ten times per 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 other week, once every three weeks, once every four weeks, or once every five weeks.

In some embodiments, the first dose is about 3.6mg and the second dose is about 7.2mg. In some embodiments, the first dose is about 3mg and the second dose is about 14mg, which is administered weekly. In some embodiments, the first dose is about 3mg and the second dose is about 7mg, which is administered weekly. In some embodiments, the first dose is about 3mg and the second dose is about 7mg, which is administered every other week.

Therapeutic method

In some embodiments, DLL3 binding proteins or DLL3 targeting trispecific proteins of the present disclosure are administered to treat neoplastic conditions. In some embodiments, the neoplastic condition is benign or malignant; solid tumors or other blood neoplasms; and in some embodiments is selected from, but not limited to: adrenal gland tumor (ADRENAL GLAND tumors), AIDS-related cancer (AIDS-associated cancers), acinar soft tissue tumor (alveolar soft part sarcoma), astrocytoma (astrocytic tumors), autonomic ganglionic tumor (autonomic ganglia tumors), bladder cancer (squamous cell carcinoma (squamous cell carcinoma) and transitional cell carcinoma (transitional cell carcinoma)) Blastula cavity disorder (blastocoelic disorders), bone cancer (bone cancer) (enamel cytoma (adamantinoma), aneurysmal bone cyst (aneurismal bone cysts), osteochondral tumor (osteochondroma), osteosarcoma (osteosarcoma)), brain and spinal cord cancer (brain AND SPINAL cord cancer), metastatic brain tumor (METASTATIC BRAIN TUMORS), and, Breast cancer (breast cancer) (including triple negative breast cancer (TRIPLE NEGATIVE breast cancer)), carotid aneurysm (carotid body tumors), cervical cancer (CERVICAL CANCER), chondrosarcoma (chondrosarcoma), chordoma (chordoma), chromorenal cell carcinoma (chromophobe RENAL CELL carpinoma), clear cell carcinoma (CLEAR CELL carpinoma), and, Colon cancer (colon cancer), colorectal cancer (colorectal cancer), benign fibrous histiocytoma of skin (cutaneous benign fibrous histiocytomas), desmoplastic small round cell tumor (desmoplastic small round cell tumors), ependymoma (ependymomas), epithelial disorder (EPITHELIAL DISORDERS), ewing's tumor (Ewing's tumors), and, Exoskeletal myxoid chondrosarcoma (extraskeletal myxoid chondrosarcoma), osteofibrogenesis deficiency (fibrogenesis imperfecta ossium), bone fibrotic dysplasia (fibrous dysplasia of the bone), gall bladder and bile duct cancer (gallbladder and bileduct cancers), gastric cancer (GASTRIC CANCER), gastrointestinal cancer (gastrointestinal), Gestational trophoblastic disease (gestational trophoblastic disease), germ cell tumor (germ cell tumors), glandular disorder (glandular disorders), head and neck cancer (HEAD AND NECK CANCERS), hypothalamic cancer (hypothalamic), intestinal cancer (INTESTINAL CANCER), islet cell tumor (ISLET CELL tumors), kaposi's Sarcoma (Kaposi's sarcomas), Renal carcinoma (KIDNEY CANCER) (nephroblastoma (nephroblastoma), papillary renal cell carcinoma (PAPILLARY RENAL CELL carpinoma)), leukemia (leukemias), lipoma/benign lipoma (lipoma/benign lipomatous tumors), liposarcoma/malignant lipoma (liposarcoma/MALIGNANT LIPOMATOUS TUMORS), liver cancer (LIVER CANCER) (hepatoblastoma (hepatoblastoma), and, Hepatocellular carcinoma (hepatocellular carcinoma)), lymphoma (lymphomas), lung cancer (lung cancer) (small cell carcinoma (SMALL CELL carcinoma), adenocarcinoma (adenocarpioma), squamous cell carcinoma (squamous cell carcinoma), large cell carcinoma (LARGE CELL carcinoma), macrophage disorder (macrophagal disorders), medulloblastoma (medulloblastoma), and the like, Melanoma (melanoma), meningioma (meningiomas), multiple endocrine adenoma (multiple endocrine neoplasia), multiple myeloma (multiple myeloma), myelodysplastic syndrome (myelodysplastic syndrome), neuroblastoma (neuroblastoma), neuroendocrine tumor (neuroendocrine tumors), ovarian cancer (ovarian cancer), ovarian cancer (B-C), Pancreatic cancer (PANCREATIC CANCERS), papillary thyroid cancer (PAPILLARY THYROID CARCINOMAS), parathyroid tumor (parathyroid tumors), pediatric cancer (PEDIATRIC CANCERS), peripheral nerve sheath tumor (PERIPHERAL NERVE SHEATH tumors), pheochromocytoma (phaeochromocytoma), pituitary tumor (pituitary tumors), Prostate cancer (prostate cancer), posterior uveal melanoma (posterious unveal melanoma), rare hematological disorders (rare hematologic disorders), renal metastasis (RENAL METASTATIC CANCER), rhabdoid tumor (rhabdoid tumor), rhabdomyosarcoma (rhabdomysarcoma), sarcoma (sarcomas), skin cancer (SKIN CANCER), and, Soft tissue sarcoma (soft-tissue sarcomas), squamous cell carcinoma (squamous CELL CANCER), gastric cancer (stomach cancer), stromal disorders (stromal disorders), synovial sarcoma (synovial sarcoma), testicular cancer (testicular cancer), thymus cancer (thymus cancer), thymoma (thymoma), thyroid metastasis cancer (thyroid METASTATIC CANCER) and uterine cancer (uterine cancer) (cervical cancer (carcinoma of the cervix), Endometrial cancer (endometrial carcinoma) and leiomyoma (leiomyoma)).

In certain embodiments, the DLL3 binding proteins or DLL3 targeted trispecific proteins of the present disclosure are used as a front line therapy and administered to a subject who has not been previously treated for cancerous conditions. In other embodiments, the DLL 3-targeted trispecific proteins of the present disclosure are used to treat subjects who have been previously treated (targeted trispecific proteins with DLL3 of the present disclosure or with other anti-cancer agents) and have relapsed or are determined to be refractory to previous treatments. In some embodiments, DLL 3-targeted trispecific proteins of the present disclosure are used to treat subjects with recurrent tumors.

In some aspects, DLL3 binding proteins or DLL3 targeting trispecific proteins of the present disclosure are administered to treat proliferative disorders including solid tumors, including but not limited to adrenal tumors, liver tumors, kidney tumors, bladder tumors, breast, stomach tumors, ovarian tumors, cervical tumors, uterine tumors, esophageal tumors, colorectal tumors, prostate tumors, pancreatic tumors, lung (small cell and non-small cell) tumors, thyroid tumors, carcinomas, sarcomas, glioblastomas, and various head and neck tumors.

In some embodiments, a DLL3 binding protein or DLL3 targeting trispecific protein of the present disclosure is administered to a subject suffering from melanoma. In some embodiments, DLL 3-targeted trispecific proteins of the present disclosure are used to diagnose, monitor, treat, or prevent melanoma. As used herein, the term "melanoma" includes all types of melanoma, including but not limited to primary melanoma (primary melanoma), malignant melanoma (MALIGNANT MELANOMA), cutaneous melanoma (cutaneous melanoma), extracutaneous melanoma (extracutaneous melanoma), superficial diffuse melanoma (superficial spreading melanoma), polypoid melanoma (polypoid melanoma), melanocyte carcinoma (melanocarcinomas), melanoma epithelium (melanoepitheliomas), melanoma (melanosarcomas), orthotopic melanoma (melanomin situ), nodular malignant melanoma (nodular malignant melanoma), freckle-like malignant melanoma (lentigo maligna melanoma), freckle-like melanoma (lentiginous melanoma), freckle-like malignant melanoma (lentiginous malignant melanoma), mucosal freckle-like melanoma (mucosal lentiginous melanoma), mucosal melanoma (mucosal melanoma), acrofreckle-like melanoma (acral lentiginous melanoma), soft tissue melanoma (soft tissue melanoma), ocular melanoma (oculmenoma), invasive melanoma (invasive melanoma), familial non-malignant melanoma (FAMILIAL ATYPICAL mole and melanoma) and focal melanoma (FAM-dysplastic melanoma) or malignant focal-2 (678).

DLL3 is a potent tumor marker expressed on many different cancers and has been found to be associated with cancer stem cells. Thus, in some embodiments, where a DLL3 binding protein or DLL3 targeting trispecific protein of the present disclosure is incorporated into a chimeric antigen receptor expressed on the lymphatic table, the resulting "DLL3 sensitized lymphocyte" (e.g., a natural killer cell or T cell that immunospecifically recognizes a DLL3 determinant) is capable of effectively eliciting an immune response against aberrant DLL3 positive cells, including cancer stem cells. This ability to effectively eliminate tumorigenic "seed" cells is often critical 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 standard of care post-treatment maintenance regimen.

More generally, a chimeric antigen receptor is an artificially constructed hybrid protein or polypeptide that contains or comprises an antigen binding domain of an antibody linked to a signaling domain (e.g., a T cell signaling or T cell activating domain). In some embodiments, a CAR comprising a DLL 3-targeted trispecific binding protein of the present disclosure has the ability to redirect the specificity and reactivity of sensitized lymphocytes (e.g., T cells) to DLL 3-positive target cells in a non-MHC-restricted manner by exploiting the antigen binding properties of an antibody or antigen binding fragment thereof. non-MHC-restricted antigen recognition enables DLL3 CAR-expressing T cells to recognize tumorigenic DLL3 independent of antigen processing, bypassing the primary mechanism of tumor escape. Furthermore, when expressed in T cells, the CAR advantageously does not dimerize with endogenous T Cell Receptor (TCR) alpha and beta chains.

In selected aspects, a DLL3 binding protein or DLL 3-targeting trispecific protein of the present disclosure is incorporated into a Chimeric Antigen Receptor (CAR), and the DLL3 CAR is administered in a CAR-based therapy effective to treat lung cancer comprising 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 cancer (LCNEC).

In some embodiments, the DLL3 binding protein or DLL3 sensitive lymphocyte is administered to a patient exhibiting a localized disease (LIMITED STAGE DISEASE) or a broad phase disease (extensive STAGE DISEASE). In other embodiments, the disclosed DLL3 targeted trispecific antibodies are administered to refractory patients (i.e., patients who relapse during or shortly after completion of the initial therapy); sensitive patients (i.e., patients who relapse more than 2-3 months after primary therapy); or a patient exhibiting resistance to a platinum-based agent (e.g., carboplatin, cisplatin, oxaliplatin) and/or a taxane (e.g., docetaxel, paclitaxel, larotaxel, or cabazitaxel). In another embodiment, the disclosed DLL3 CAR treatments are effective for treating ovarian cancer, including ovarian serous carcinoma (ovarian-serous carcinoma) and ovarian papillary serous carcinoma (ovarian-PAPILLARY SEROUS CARCINOMA).

In some embodiments, the disclosed DLL3 binding proteins or DLL3 targeted trispecific binding proteins are used to prevent, treat, or diagnose tumors with neuroendocrine characteristics or phenotypes, including neuroendocrine tumors. True or typical neuroendocrine tumors (NET) produced by the decentralized endocrine system are relatively rare, occurring at 2-5 cases per 100,000 individuals, but with a high degree of progressive (progressive). Neuroendocrine tumors occur in the kidney, genitourinary tract (bladder, prostate, ovary, cervix and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma (medullary thyroid cancer)) and lung (small cell lung cancer and large cell neuroendocrine carcinoma). These tumors may secrete a variety of hormones, including serotonin and/or chromogranin a, which may cause debilitating symptoms known as carcinoid syndrome. Such tumors may be represented by positive immunohistochemical markers, such as neuronal specific enolase (NSE, also known as gamma enolase, gene symbol = ENO 2), CD56 (or NCAM 1), chromogranin a (CHGA) and Synaptobrevin (SYP), or by genes known to exhibit increased expression, such as ASCL1. Traditional chemotherapy is not particularly effective in treating neuroendocrine tumors, and liver metastases are a common outcome. In some embodiments, the disclosed DLL 3-targeted trispecific antibodies are advantageously used in the treatment of neuroendocrine tumors, and in some embodiments, they are used in the treatment, prevention, or diagnosis of pseudoneuroendocrine tumors that mimic, resemble, or exhibit common traits with typical neuroendocrine tumors in genotype or phenotype (pNET). Pseudoneuroendocrine tumors or tumors with neuroendocrine characteristics are tumors produced by cells of the diffuse neuroendocrine system or cells in which the neuroendocrine differentiation cascade is abnormally reactivated during oncogenic processes. Such pNET typically have certain phenotypic or biochemical characteristics, including the ability to produce a subset of bioactive amines, neurotransmitters, and peptide hormones, as compared to the traditionally defined neuroendocrine tumor. Histologically, such tumors (NET and pNET) have a common appearance, which generally show densely connected small cells, with minimal cytopathogenicity and round to oval spotted nuclei. In some embodiments of the present disclosure, commonly expressed histological or genetic markers used to define neuroendocrine and pseudoneuroendocrine tumors include, but are not limited to, chromogranin A, CD, synaptobrevin, PGP9.5, ASCL1, and neuron-specific enolase (NSE). Thus, in some embodiments, DLL 3-targeted trispecific proteins, DLL3 CARs, or DLL 3-sensitized lymphocytes of the present disclosure, or any combination thereof, are advantageously used to treat pseudo-and classical neuroendocrine tumors, such as neuroendocrine tumors (NET and pNET) arising in the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid cancer), and lung (small cell lung cancer and large cell neuroendocrine cancer). Furthermore, in some embodiments, DLL3 of the present disclosure targets a trispecific protein, DLL3 CAR, or DLL3 sensitized lymphocyte, or any combination thereof, for use in treating a tumor that expresses one or more markers, such as NSE, CD56, synaptobrevin, chromogranin A, ASCL1, or PGP9.5 (UCHL 1). In some embodiments, DLL3 of the present disclosure targets a trispecific protein, DLL3 CAR, or DLL3 sensitized lymphocyte, or any combination thereof, for use in treating a subject having a tumor of nse+ or cd56+ or pgp9.5+ or ASCL + or syp+ or chga+ or any combination thereof.

In another embodiment, DLL3 of the present disclosure targets a trispecific protein, DLL3CAR, or DLL3 sensitized lymphocyte, or any combination thereof, for maintenance therapy to reduce or eliminate the chance of tumor recurrence after initial manifestation of the disease. In some cases, the condition has been treated and the original tumor mass has been eliminated, reduced or otherwise ameliorated so that the patient is asymptomatic or in remission. At this point, a pharmaceutically effective amount of the disclosed DLL3 binding proteins, DLL3 targeted trispecific proteins of the present disclosure, DLL3 CARs, or DLL3 sensitized lymphocytes, or any combination thereof, is administered to the subject one or more times, whether using standard diagnostic procedures, with or without disease indication. In some embodiments, DLL 3-targeted trispecific proteins, DLL3 CARs, or DLL 3-sensitized lymphocytes of the present disclosure, or any combination thereof, are administered on a regular schedule over a period of time, e.g., weekly, biweekly, monthly, six weeks, bi-monthly, tri-monthly, six months, or yearly, e.g., to reduce the likelihood of disease recurrence. Furthermore, in some embodiments, such treatment continues for weeks, months, years, or even indefinitely, depending on patient response and clinical and diagnostic parameters.

In another embodiment, the DLL3 binding protein, DLL3 targeting trispecific protein of the present disclosure, DLL3 CAR, or DLL3 sensitized lymphocyte, or any combination thereof, is used prophylactically or as an adjuvant therapy to prevent or reduce the likelihood of tumor metastasis following a oncologic reduction procedure (debulking procedure). As used in this disclosure, "oncological operation" is defined broadly and means any operation, technique, or method that eliminates, reduces, treats, or improves a tumor or tumor proliferation. Exemplary oncological procedures include, but are not limited to, surgery, radiation therapy (i.e., beam radiation), chemotherapy, immunotherapy, or ablation. In some embodiments, at an appropriate time, the DLL3 binding protein, the DLL3 targeting trispecific protein of the present disclosure, the DLL3 CAR, or the DLL3 sensitized lymphocyte, or any combination thereof, is administered as suggested by a clinical, diagnostic, or theranostic procedure to reduce tumor metastasis. In some embodiments, the dosing regimen is accompanied by appropriate diagnostic or monitoring techniques that allow modifications to it.

Other embodiments of the present disclosure include administering a DLL3 binding protein, a DLL3 targeted trispecific protein of the present disclosure, a DLL3 CAR, or a DLL3 sensitized lymphocyte, or any combination thereof, to a subject that is asymptomatic but at risk of developing a proliferative disorder. That is, in some embodiments, DLL3 binding proteins, DLL 3-targeted trispecific proteins of the present disclosure, DLL3 CARs, or DLL 3-sensitized lymphocytes, or any combination thereof, are used in a prophylactic sense and administered to a patient who has been examined or tested and has one or more noted risk factors (e.g., genomic indications, family history, in vivo or in vitro test results, etc.) alone not yet developing neoplasia. In such cases, one skilled in the art will be able to determine an effective dosing regimen through empirical observations or through accepted clinical practices.

As used herein, in some embodiments, the term "treatment" refers to therapeutic treatment in which the purpose is to slow down (alleviate) an undesired physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. For purposes described herein, beneficial or desired clinical results include, but are not limited to: alleviation of symptoms; reduced extent of the condition, disorder or disease; the state of the condition, disorder or disease is stable (i.e., not worsening); delay in onset or slowing progression of a condition, disorder or disease; an improvement in a condition, disorder or disease state; and the relief (whether partial or total) (whether detectable or undetectable) or enhancement or amelioration of a condition, disorder or disease. Treatment involves eliciting a clinically significant response without undue levels of side effects. Treatment also includes extending survival compared to the expected survival without treatment. In other embodiments, "treatment" or "treatment" refers to a prophylactic measure in which the purpose is to delay the onset or reduce the severity of an undesired physiological condition, disorder or disease, e.g., a person susceptible to a disease (e.g., an individual carrying a genetic marker for a disease such as breast cancer).

In some embodiments of the methods described herein, a DLL3 binding protein, DLL3 targeting trispecific protein, or composition as described herein is administered in combination with an agent for treating a particular disease, disorder, or condition. Agents include, but are not limited to, therapies involving: therapies involving antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiation therapies (directed delivery of gamma rays, X-rays, and/or radioisotopes, microwaves, UV radiation, etc.), gene therapies (e.g., antisense, retroviral therapies, etc.), and other immunotherapies. In some embodiments, an anti-DLL 3 binding protein or an anti-DLL 3 targeting trispecific protein as described herein is administered in combination with an antidiarrheal agent, an antiemetic agent, an analgesic, an opioid, and/or a non-steroidal anti-inflammatory agent. In some embodiments, an anti-DLL 3 binding protein or an anti-DLL 3 targeting trispecific protein as described herein is administered in combination with an anti-cancer agent. Non-limiting examples of anticancer agents useful in various embodiments of the present disclosure (including pharmaceutical compositions and dosage forms and kits of the present disclosure) include: acitretin; doxorubicin; albendazole hydrochloride; dyclonine; aldolizhen; aldesleukin; altretamine; an Bomei elements; amitraz acetate; aminoglutethimide; amsacrine; anastrozole; an aflatoxin; asparaginase; qu Linjun elements; azacitidine; azatepa; dorzolomycin; BAMASITANG; bentepa; bicalutamide; hydrochloride acid bisantrene; binaford; the comparison is newer; bleomycin sulfate; sodium buconazole; bromopirimin; busulfan; actinomycin C; a card Lu Gaotong; carpronium chloride; a card Bei Tim; carboplatin; carmustine; carminomycin hydrochloride; the card is folded for new use; sidefagon; chlorambucil; sirolimus; cisplatin; cladribine; klebsiella mesylate; cyclophosphamide; cytarabine; dacarbazine; actinomycin D; daunorubicin hydrochloride; decitabine; right omaboplatin; deazaguanning; debezaguanine mesylate; deaquinone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; drotaandrosterone propionate; daptomycin; eda traxas; epoxicillin hydrochloride; elsamitrucin; enlobaplatin; enpramine ester; epiridine; epirubicin hydrochloride; erbzol; exenatide hydrochloride; estramustine; estramustine sodium phosphate; itraconazole; etoposide; etoposide phosphate; ai Tuobo Ning; hydrochloric acid process Qu; fazab; fenretinide; fluorouridine; fludarabine phosphate; fluorouracil; flucitabine; a phosphoquinolone; fosetrexed sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; tamofosin; interleukin II (including recombinant interleukin II or rIL 2); interferon alpha-2 a; interferon alpha-2 b; interferon alpha-n 1; interferon alpha-n 3; interferon beta-I a; interferon gamma-I b; platinum isopropoxide; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprorelin acetate; liazole hydrochloride; lomefen Qu Suona; lomustine; losoxanone hydrochloride; maxolol; maytansine; nitrogen mustard hydrochloride; megestrol acetate; melengestrol acetate; melphalan; minoxidil; mercaptopurine; methotrexate; methotrexate sodium; chlorphenidine; metrotifer; rice Ding Duan; mitomycin (mitocarcin); rice tray Kong Xing (mitocromin); mitoJielin; mitomycin; mitomycin; mitopristal culture; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; norgamycin; oxaliplatin; an oxy Shu Lun; paclitaxel; cultivating an asparate; pelimycin; pentose mustard; pelomycin sulfate; pesphosphamide; pipobromine; piposulfan; pyri Luo Enkun hydrochloride; plicamycin; pralometan; porphin sodium; pofemycin; prednisomustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazolofuranomycin; lipoadenosine; rogestini; sha Fenge; hydrochloric acid Sha Fenge; semustine; xin Quqin; sodium spp; rapamycin; spiral germanium hydrochloride; spiromustine; spiroplatinum; streptozotocin; streptozotocin; sulfochlorphenylurea; tarithromycin; sodium tecogalan; tegafur; tilonthraquinone hydrochloride; temoporphine; teniposide; luo Xilong; testosterone lactone; thioazane; thioguanine; thiotepa; thiazole carboxamide nucleosides; tirapazamine; toremifene citrate; tramadol acetate; troxirebaudil phosphate; trimetha sand; triclosan glucuronate; triptorelin; tobrachlorazole hydrochloride; uramustine; uretidine; vaptan; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinblastine sulfate; vinpocetine sulfate; vincristine sulfate; vinorelbine tartrate; vinorelbine sulfate; vinblastidine sulfate; fucloxazole; platinum; clean stastatin; zorubicin hydrochloride. Other examples of anticancer agents include, but are not limited to: 20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyl uracil; abiraterone; doxorubicin; acyl fulvenes; adenosine cyclopentanol; aldolizhen; aldesleukin; ALL-TK antagonists; altretamine; amoustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; an angiogenesis inhibitor; antagonist D; antagonist G; an Leili grams; anti-dorsal morphogenic protein-1 (anti-dorsalizing morphogenetic protein-1); prostate cancer antiandrogens; antiestrogens; anti-neoplastic ketones; an antisense oligonucleotide; glycine Afedimycin; apoptosis gene modulators; apoptosis modulators; no purine acids; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; altamitant; amoustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azalide; diazo amino acids; baccatin III derivatives; balanol; BAMASITANG; BCR/ABL antagonists; benzodihydropamoic acid; benzoyl staurosporine; beta-lactam derivatives; beta-alethine; beta clamycin B; betulinic acid; bFGF inhibitors; bicalutamide; a specific group; biaziridinyl spermine; binaford; double Qu Qun A (bistratene A); the comparison is newer; breflate; bromopirimin; pudding; butyl thioamino acid sulfoxide imine; calcipotriol; calpain C; camptothecin derivatives; canary pox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; caRest M3; CARN 700,700; cartilage derivative inhibitors; the card is folded for new use; casein kinase Inhibitors (ICOS); chestnut tree spermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cilazaprost; cis-porphyrin; cladribine; clomiphene analogs; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogs; conagenin; crambescidin 816; klebsiella; nostoc 8; nostoc a derivatives; curacin a; cyclopentaanthraquinone; a cycloplatin (cycloplatam); epirubicin; sodium cytarabine phosphate; a cytolytic factor; hexane estrol phosphate (cytostatin); dacliximab; decitabine; dehydromembranous ecteinascidin B; desertraline; dexamethasone; right ifosfamide; dexrazoxane; right verapamil; deaquinone; ecteinascidin B; didox; diethyl norspermine; dihydro-5-azacytidine; 9-dihydrotaxol; dioxamycomycin (dioxamycin); diphenyl spiromustine; docetaxel; twenty glycol; dolasetron; deoxyfluorouridine; droloxifene; dronabinol; dacarbazine SA; ebselen; icotemustine; edefloxin; edestin (edrecolomab); ornithine difluoride; elemene; bupirimate; epirubicin; eplerenone; estramustine analogues; an estrogen agonist; estrogen antagonists; itraconazole; etoposide phosphate; exemestane; fatrazole; fazab; fenretinide; febuxostat; finasteride; fraapine degree; fluodosteine; fluasterone; fludarabine; fluorodaunorubicin hydrochloride (fluorodaunorunicin hydrochloride); fomesalamine; fumesteine; fosetrexed; fotemustine; gadolinium germanorphyrin (gadolinium texaphyrin); gallium nitrate; gaboxacitabine; ganirelix; a gelatinase inhibitor; gemcitabine; glutathione inhibitors; hepsulfam; regulating protein; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; iblock Meng Tong; tamofosin; ilomastat; imidazo acridone; imiquimod; an immunostimulatory peptide; insulin-like growth factor-1 receptor inhibitors; an interferon agonist; an interferon; interleukins; iodobenzyl guanidine; iodinated doxorubicin; 4-sweet potato bittering alcohol; luo Pula; eostiradin; a foreign country grazole; different high halichondrin B; itasetron; gas pranoprofen (jasplakinolide); card Ha Lali and F (kahalalide F); lamellarin-N triacetate; lanreotide; rapamycin; leiging pavilion; lentinan sulfate; lei Bosi statins; letrozole; leukemia inhibitory factor; leukocyte interferon-alpha; leuprorelin + estrogen + progesterone; leuprorelin; levamisole; lidazole; linear polyamine analogs; a lipophilic disaccharide peptide; a lipophilic platinum compound; lissoclinamide 7 a 7; lobaplatin; earthworm phospholipids; lometrexed; lonidamine; losoxantrone; HMG-CoA reductase inhibitors (such as, but not limited to, lovastatin, pravastatin, fluvastatin, statins, simvastatin, and atorvastatin); loxoribine; lurtoltecan; lutetium texaphyrin; lysofylline; cleaving the peptide; maytansine; manostatin a; marimastat; maxolol; mammary gland silk-screen protein; matrix dissolution factor inhibitors; matrix metalloproteinase inhibitors; minoxidil; meprobamate (merbarone); avorelin; methioninase (methioninase); weifuan; MIF inhibitors; mifepristone; miltefosine; midirtine; mismatched double stranded RNA; mitoguazone; dibromodulcitol; mitomycin analogs; mitonaphthylamine; mitoxin fibroblast growth factor-saporin; mitoxantrone; mo Faluo a material; moraxetin; a human chorionic gonadotrophin monoclonal antibody; monophosphoryl lipid a+ mycobacterial cell wall sk; mo Pai darol; a multi-drug resistance gene inhibitor; therapy based on multiple tumor suppressor gene 1; an anticancer agent of mustard; indian sponge B (mycaperoxide B); mycobacterial cell wall extracts; myriaporone; n-acetyldinaline; n-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; natto pavilion; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidases; nilutamide; nisamycin; nitric oxide modulators; a nitroxide antioxidant; nitrullyn; o6-benzyl guanine; octreotide; okicenone; an oligonucleotide; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducers; oxaliplatin; austenite Sha Telong; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronate; panaxatriol; panomifene; parabactin; poisson Puding; cultivating an asparate; pefloxacin (peldesine); sodium pentose polysulfate; prastatin; spray trazodone; perfluorobromoalkane; pesphosphamide; perillyl alcohol; phenazinomycin (phenazinomycin); phenylacetate; a phosphatase inhibitor; a streptolysin preparation (picibanil); pilocarpine hydrochloride; pirarubicin; pitroxine; PLACETIN A; placetin B; a plasminogen activator inhibitor; a platinum complex; a platinum compound; platinum-triamine complexes; porphin sodium; laver mycin; prednisone; propyl bisacridone; prostaglandin J2; a proteasome inhibitor; protein a-based immunomodulators; protein kinase C inhibitors; microalgae protein kinase C inhibitor; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; rhodopsin; methoxy pyrazoline acridine; pyridoxylated hemoglobin polyoxyethylene conjugates; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; demethylated reteplatin; rhenium Re186 etidronate; rhizopus extract; ribozymes; RII isotretinoin amide; rogestini; roxitoxine; romidepsin; luo Kuimei g; rubiginone B1; ruboxyl; sha Fenge; saintopin; sarCNU; sarcophytol A; a sauce pavilion; sdi 1 mimetic; semustine; age-derived inhibitor 1; a sense oligonucleotide; a signal transduction inhibitor; a signal transduction modulator; a single chain antigen binding protein; a sirzopyran; sobuzocine; sodium boron carbazate; sodium phenylacetate; solverol; a growth regulator binding protein; soxhaustmine; spanish acid; spica D (spicamycin D); spiromustine; stoneley Pan Ding; sponge chalone 1 (spongistatin 1); squalamine; stem cell inhibitors; stem cell division inhibitors; stipiamide; a matrix-dissolving protease inhibitor; sulfinosine; potent vasoactive intestinal peptide antagonists; suradista; suramin; swainsonine; a synthetic glycosaminoglycan; tamustine; tamoxifen methyl iodide; niu Huangmo statin; tazarotene; sodium tecogalan; tegafur; tellurapyrylium; telomerase inhibitors; temoporphine; temozolomide; teniposide; tetrachlorodecaoxide (tetrachlorodecaoxide); tetrazomine; mycoembryoin (thaliblastine); thiocoraline (thiocoraline); thrombopoietin; thrombopoietin mimetics; thymalfasin; an agonist of the thymic hormone receptor; thymic treonam; thyroid stimulating hormone; ethyltin protorhodopsin (tinethyl etiopurpurin); tirapazamine; titanocene dichloride (titanocene bichloride); topsentin; toremifene; totipotent stem cell factor; a translation inhibitor; tretinoin; triacetyl uridine; troxirebaudibine; trimetha sand; triptorelin; tropisetron; tolofaciron; tyrosine kinase inhibitors; tyrosine phosphorylation inhibitor (tyrphostin); UBC inhibitors; ubenimex; a urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vaptan; variolin B; a erythrocyte gene therapy vector system; venlafaxine; veratramine; verdins; verteporfin; vinorelbine; weikesaitin (vinxaltine); Vorozole; zanotarone; platinum; benzylidene vitamin C (zilasorb); and clean stats Ding Sizhi. Additional anticancer drugs are 5-fluorouracil and folinic acid. Both agents are particularly useful when used in methods employing thalidomide and topoisomerase inhibitors. In some embodiments, the anti-DLL 3 targeted trispecific proteins of the present disclosure are used in combination with gemcitabine. In some embodiments, the DLL3 targeted trispecific protein as described herein is administered before, during, or after surgery.

Method for detecting DLL3 expression and diagnosis of DLL 3-related cancers

According to another embodiment of the present disclosure, a kit for detecting DLL3 expression in vitro or in vivo is provided. Kits include the aforementioned DLL3 binding proteins, DLL3 targeted trispecific proteins (e.g., trispecific proteins containing a labeled anti-DLL 3 single domain antibody or 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 enzymatic label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

In some cases, DLL3 expression is detected in a biological sample. The sample may be any sample including, but not limited to, tissue from biopsies, autopsies and pathological specimens. Biological samples also include tissue sections, e.g., frozen sections for histological purposes. Biological samples also include body fluids such as blood, serum, plasma, sputum, spinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates.

In one embodiment, a method of determining whether a subject has cancer by: contacting a sample from the subject with an anti-DLL 3 single domain antibody or an anti-DLL 3 trispecific protein disclosed herein; and detecting binding of the single domain antibody to the sample. An increased binding of the antibody to the sample as compared to the binding of the antibody to the control sample identifies the subject as suffering from cancer.

In another embodiment, a method of confirming a cancer diagnosis in a subject by: contacting a sample from a subject diagnosed with cancer with an anti-DLL 3 single domain antibody or an anti-DLL 3 trispecific protein disclosed herein; and detecting binding of the antibody to the sample. The increased binding of the antibody to the sample as compared to the binding of the antibody to the control sample confirms a diagnosis of cancer in the subject.

In some examples of the disclosed methods, DLL3 binding single domain antibodies to DLL3 binding proteins or trispecific proteins are directly labeled. In some examples, the method further comprises contacting a sample with a secondary antibody that specifically binds to an anti-DLL 3 single domain antibody or an anti-DLL 3 trispecific protein; and detecting binding of the secondary antibody. The binding of the secondary antibody to the sample increases the detection of cancer in the subject or the confirmation of cancer diagnosis in the subject as compared to the binding of the secondary antibody to the control sample. In some cases, the cancer is neuroendocrine, prostate, lung, gastric, squamous cell, pancreatic, cholangiocarcinoma, triple negative breast or ovarian cancer, or any other type of cancer that expresses DLL 3. In some examples, the control sample is a sample from a subject without cancer. In a particular example, the sample is a blood or tissue sample.

In some cases, antibodies that bind (e.g., specifically bind) DLL3 are directly labeled with a detectable label. In another embodiment, the antibody (primary antibody) that binds (e.g., specifically binds) DLL3 is unlabeled, and the secondary antibody or other molecule that can bind the antibody that specifically binds DLL3 is labeled. The secondary antibody is selected such that it is capable of specifically binding to a specific species and class of primary antibodies. For example, if the primary antibody is a llama IgG, the secondary antibody may be an anti-llama IgG. Other molecules that may 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 secondary antibodies are described above and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic reagents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-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 isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic reagent is gadolinium and a non-limiting exemplary radiolabel includes 125I, 131I, 35S, or 3H.

In an alternative embodiment, DLL3 can be analyzed 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 DLL3. In this assay, a biological sample, a labeled DLL3 standard, and an antibody that specifically binds DLL3 are combined together and the amount of labeled DLL3 standard that binds to unlabeled antibody is determined. The amount of DLL3 in a biological sample is inversely proportional to the amount of labeled DLL3 standard that binds to an antibody that specifically binds DLL3.

The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, antibodies that specifically bind DLL3 can be used to detect DLL3 production in cells in a cell culture. In another embodiment, the antibodies 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, DLL3 is cell surface DLL3. In other examples, DLL3 is soluble DLL3 (e.g., DLL3 in a cell culture supernatant or soluble DLL3 in a body fluid sample such as a blood or serum sample).

In one embodiment, a kit for detecting DLL3 in a biological sample, such as a blood sample or a tissue sample, is provided. For example, to confirm a cancer diagnosis of a subject, a biopsy may be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample may be obtained to detect the presence of soluble DLL3 protein or fragment. In accordance with the present disclosure, a kit for detecting a polypeptide generally comprises a single domain antibody that specifically binds DLL 3. In some embodiments, an antibody fragment, such as an scFv fragment, VH domain, or Fab, is included in the kit. In further embodiments, the antibody is labeled (e.g., with fluorescence, radioactivity, or an enzyme).

In one embodiment, the kit includes instructional materials instructing the use of a means for binding antibodies to DLL 3. The instructional material may be written in electronic form (such as a computer diskette or optical disk) or may be visual (such as a video file), or may be provided over an electronic network, such as the Internet, world Wide Web, intranet, or other network. The kit may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally comprise means for detecting the label (such as an enzyme substrate for enzyme labelling, a filter set for detecting fluorescent labels, a suitable second label such as a secondary antibody, etc.). The kit may additionally include buffers and other reagents conventionally used to carry out particular methods. Such kits and suitable contents are well known to those skilled in the art.

In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassay may vary with the particular format employed, the method of detecting DLL3 in a biological sample generally includes the step of contacting the biological sample with an antibody that specifically reacts with a DLL3 polypeptide under immunoreactive conditions. Antibodies are specifically bound under immunoreactive conditions to form an immunocomplex, and the presence of the immunocomplex (bound antibody) is detected directly or indirectly.

Methods for determining the presence or absence of cell surface markers are well known in the art. For example, the antibodies may 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 may also be used in immunoassays such as, but not limited to, radioimmunoassays (RIA), ELISA, or immunohistochemical assays. Antibodies can also be used for Fluorescence Activated Cell Sorting (FACS). FACS employs multiple color channels, low angle and obtuse angle light scattering detection channels, and impedance channels at other more complex detection levels to separate or sort cells (see us patent No. 5,061,620). As disclosed herein, any single domain antibody that binds DLL3 can be used in these assays. Thus, antibodies may be used in conventional immunoassays, including but not limited to ELISA, RIA, FACS, histoimmunohistochemistry, western blotting, or immunoprecipitation.

Examples

Example 1: screening phage display libraries to identify DLL3 binding domains

Llama was immunized with purified DLL3 protein expressed in EXPI293 ^TM cells. Phage display libraries for expression of heavy chain variable antibody domains were constructed from circulating B cells (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 produced a signal in an ELISA screen (SEQ ID No.1 to SEQ ID No. 52). The CDR1, CDR2 and CDR3 sequences of these heavy chain variable domains are SEQ ID nos. 443 to 494, 885 to 936 and 1327 to 1378, respectively.

Example 2: humanized and T cell dependent cytotoxicity assays for DLL3 binding to single domain antibodies

Thirty-four (SEQ ID No.53 to SEQ ID No. 86) exemplary llama anti-DLL 3 heavy chain-only single domain antibodies from example 1 were humanized. The CDR1, CDR2 and CDR3 sequences of the 34 heavy chain-only single domain antibodies were SEQ ID nos. 495 to 528, 937 to 970 and 1379 to 1412, respectively.

The humanized anti-DLL 3 sequence was cloned into an expression vector in the form of an expression construct comprising a signal domain followed by the variable domain of the heavy chain only of anti-DLL 3, followed by a GGGGSGGGS linker (SEQ ID No. 1808), followed by an anti-human albumin single domain antibody 10G (SEQ ID No. 1774), followed by a GGGGSGGGS linker (SEQ ID No. 1808), followed by an anti-human CD3 antibody 2B2 (SEQ ID No. 1793), followed by a HHHHHH tag (SEQ ID No. 1819) to generate an anti-DLL 3 trispecific construct.

The anti-DLL 3 trispecific construct containing the humanized anti-DLL 3 binding sequence was then transfected into EXPI293 ^TM cells. These anti-DLL 3 trispecific constructs were engineered with protein a binding sites and the amount of anti-DLL 3 trispecific construct in conditioned medium from transfected EXPI293 ^TM cells was quantified using an Octet instrument with a protein a tip. A trispecific protein having a molecular weight similar to that of the anti-DLL 3 trispecific protein was used as a standard.

The binding affinity of anti-DLL 3 trispecific proteins to human and cynomolgus monkey DLL3 proteins was measured using a conditioned medium containing a known concentration of anti-DLL 3 trispecific proteins using a method of expressing DLL3 proteins as human IgG1-Fc fusions, and the measurement was performed using an Octet instrument with an anti-human Fc tip. K _D measurements were performed using a single 50nM concentration of anti-DLL 3 trispecific protein, allowing sequencing based on potency. The relative affinities measured as described above are listed in table 1. All sequences were found to bind human DLL3 with a relative affinity (K _D) in the range of 0.5 to 42nM. Some sequences were found to bind cynomolgus DLL3 with similar affinities to human DLL3, and the relative affinities of those sequences binding to cynomolgus DLL3 are also shown in table 1.

Conditioned medium was also tested in a T cell dependent cytotoxicity assay (see Nazarian AA, archibeque IL, nguyen YH, wang P, SINCLAIR AM, POWERSDA.2015.J Biomol Screen.20:519-27). In this assay, luciferase-labeled DMS-153 cells (small cell lung carcinoma cell line; ATCC number)CRL-2064 ^TM) was combined with purified human T cells from a donor and a titration of the tested anti-DLL 3 trispecific protein.

It is hypothesized that if the anti-DLL 3 trispecific protein directs the T-cells to kill DLL3 expressing DMS-153 cells, the viability of the DMS-153 cells should be reduced as determined by the luciferase assay 48 hours after the start of the experiment.

As shown in fig. 2-6, which show graphs of representative TDCC data, several exemplary anti-DLL 3 trispecific proteins were able to reduce the viability of DMS-153 cells. FIG. 2 shows the results of TDCC assays for anti-DLL 3 trispecific proteins comprising the DLL3 binding domains DH18 (SEQ ID No. 59), DH11 (SEQ ID No. 55), DH67 (SEQ ID No. 42) and DH56 (SEQ ID No. 73). FIG. 3 shows the results of TDCC assays for anti-DLL 3 trispecific proteins comprising the DLL3 binding domains DH2 (SEQ ID No. 60), DH43 (SEQ ID No. 68), DH10 (SEQ ID No. 54) and DH6 (SEQ ID No. 75). FIG. 4 shows the results of TDCC assays for DLL3 trispecific proteins comprising the DLL3 binding domains DH82 (SEQ ID No. 81), DH23 (SEQ ID No. 62), DH89 (SEQ ID No. 84) and DH17 (SEQ ID No. 58). FIG. 5 shows the results of TDCC assays for DLL3 trispecific proteins comprising the DLL3 binding domains DH83 (SEQ ID No. 82), DH12 (SEQ ID No. 56), DH61 (SEQ ID No. 76) and DH29 (SEQ ID No. 64). FIG. 6 shows the results of TDCC assays for DLL3 trispecific proteins comprising the DLL3 binding domains DH58 (SEQ ID No. 74) and DH70 (SEQ ID No. 79). The negative control for TDCC assay was a trispecific protein targeting GFP instead of DLL3 (as shown in figure 6), which did not direct T cells to kill DMS-153 cells. EC ₅₀ values determined at TDCC are also listed in table 1. These values range from 69pM to 11nM.

Table 1: the activity of humanized anti-DLL 3 trispecific proteins in the DMS-153TDCC assay and their affinity for human and cynomolgus monkey DLL3 proteins. K _D measurements were performed using a single concentration of anti-DLL 3 trispecific protein. TDCC assays were performed using human T cells. n/d indicates that no binding was detected.

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Example 3: using two humanized DLL3 single domain antibodies from previous examples, phage display libraries were screened to identify DLL3 binding domains with higher binding affinities

Two humanized antibody sequences DH43 (SEQ ID No. 68) and DH6 (SEQ ID No. 75) were used as starting points for the preparation of phage display libraries (according to the method as described in WO2016187101A 2). The anti-DLL 3 sequences from this panning were then cloned into expression vectors in the form of expression constructs comprising a signal domain followed by the variable domain of the heavy chain only of anti-DLL 3, 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 anti-DLL 3 trispecific proteins. These constructs were transfected into EXPI293 ^TM cells and the expressed anti-DLL 3 trispecific proteins were quantified as described in example 2. The sequences of the clones identified from panning were SEQ ID No.87 to SEQ ID No.367. Table 2 provides CDR variations obtained in DH43 DLL3 conjugate sequences after phage display selection. Three of the clones identified from panning were engineered for SEQ ID nos. 199 (2E 05), 330 (4D 09), and 365 (4H 011) to generate variants, each having a single amino acid change from the parent sequence, e.g., to remove potential metabolic tendencies of the parent sequence (metabolic liabilities). Specifically, an engineered variant comprising the DLL3 binding domain of SEQ ID No.227 (2E 05-M106Y), SEQ ID No.228 (2E 05-M106Q) as SEQ ID No.199 (2E 05); an engineered variant comprising DLL3 binding domain of SEQ ID No.366 (4D 09-M34L) SEQ ID No.330 (4D 09); and comprises an engineered variant of SEQ ID No.367 (4H 11-M34L) with the DLL3 binding domain of SEQ ID No.365 (4H 011). The CDR1 sequences of these DLL3 binding clones identified by panning were SEQ ID nos. 529 to 809, the CDR2 sequences of the clones identified by panning were SEQ ID nos. 971 to 1251, and the CDR3 sequences of the clones identified by panning were SEQ ID nos. 1413 to 1691.

Table 2: variants of CDR sequences according to the amino acid positions of DH43 and derivatives thereof

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The binding affinity of the anti-DLL 3 trispecific protein to human DLL3 protein was measured using a method of expressing the biotinylated version of the human DLL3 protein as a human IgG1 fusion protein using a conditioned medium with a known concentration of the anti-DLL 3 trispecific protein, and the binding affinity measurement was performed in an Octet instrument with a streptavidin tip. K _D measurements were performed using a single 50nM concentration of anti-DLL 3 trispecific protein, thereby achieving potency sequencing. In this experiment, the relative K _D values of affinity matured clones ranged from 2.3nM to 64nM, as listed in Table 3. Based on four conditioned medium samples from four transfections, the K _D values for the parental conjugates DH43 and DH6 were 7.7.+ -. 0.6nM and 9.9.+ -. 0.3nM, respectively.

For selected DLL3 conjugate molecules identified in this round of panning and for parent DLL3 conjugates DH43 and DH6, more accurate affinity measurements for human DLL3 were made using anti-DLL 3 trispecific proteins at concentrations of 60nM, 20nM, 6.67nM, and 2.22 nM. Furthermore, relative affinity measurements were performed using only 60nM of anti-DLL 3 trispecific protein. Binding affinities determined from more accurate measurements of certain anti-DLL 3 binding molecules are listed in table 4 at [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 _D value of 8.9nM, while the highest affinity progeny molecule 1H012 (SEQ ID No. 162) had an affinity of 2.9nM. In addition, 1H012 (SEQ ID No. 162) also retained the ability to bind cynomolgus monkey DLL 3. Also in this study, the parent binder DH6 had a K _D value of 9.0nM, while the highest affinity progeny molecule 4H011 (SEQ ID No. 365) had an affinity of 3.9nM. In addition, 4H011 (SEQ ID No. 365) also retains the ability to bind to cynomolgus monkey DLL 3.

Twenty-two DLL3 conjugate molecules identified in this round of panning were selected for testing in TDCC assays with DMS-153 cells using the same protocols as described in example 2. Exemplary TDCC data are plotted in the form of graphs in fig. 7-11, and a summary of EC ₅₀ values is set forth in table 5. In this experiment, the parent DLL3 molecules DH43 and DH6 had EC ₅₀ values of 200nM and 340nM, respectively. The most potent progeny molecule of DH43 was 1H012 (SEQ ID No. 162) with an EC ₅₀ value of 28nM, indicating a greater than 7-fold increase in TDCC potency compared to the parent DLL3 conjugate DH 43. The most potent progeny molecule of DH6 is 4H011 (SEQ ID No. 365) with an EC ₅₀ value of 36nM, thus showing a greater than 8-fold increase in TDCC potency compared to the parent DLL3 conjugate molecule. The GFP-targeting control trispecific protein used as a control was inactive in this assay (as shown in fig. 11).

Table 3: relative affinity against DLL3 trispecific proteins

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Table 4: binding constants to human DLL3 determined using three different concentrations of anti-DLL 3 trispecific proteins and to cynomolgus monkey DLL3 determined using a single concentration of anti-DLL 3 trispecific proteins

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Table 5: DMS-153TDCC values for affinity matured anti-DLL 3 trispecific proteins in conditioned medium using triplicates of human T cells

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Example 4: cloning of selected DLL3 binding molecules from example 3 into mammalian cells

The anti-DLL 3 trispecific protein described in example 3, as well as the parental DLL3 conjugate molecule, were subcloned into CHO cell expression vectors and stably transfected in CHO cells (see Running Deer and Allison 2004.Biotechnol. Prog. 20:880-889). DLL3 conjugate molecule ：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)( is also referred to herein as 1H 12); 2E05 (SEQ ID No. 199); 2H02 (SEQ ID No. 221); 4D011 (SEQ ID No. 332) (also referred to herein as 4D 11); 2E02 (SEQ ID No. 198); 4H11-M34L (SEQ ID No. 367); 1A011 (SEQ ID No. 95) (also referred to herein as 1A 11); DH6 (SEQ ID No. 75); and DH43 (SEQ ID No. 68). After expression in CHO cells, the anti-DLL 3 trispecific proteins were purified from the stable clone pool in conditioned medium using protein a and ion exchange chromatography. Using the same method as described in example 2, the purified protein was tested in TDCC assay. EC ₅₀ values determined for TDCC of this example are listed in table 6 and graphs of the data are shown in fig. 12-15. The most potent molecule 2E05-M106Q (SEQ ID No. 228) has an EC ₅₀ value of 41nM, which is 6.6-fold more potent than the parent molecule DH 43. The most potent molecule derived from DH6 is 4D09-M34L (SEQ ID No. 366) with an EC ₅₀ value of 54nM and 4.4-fold greater effectiveness than the parent molecule DH 6.

Table 6: TDCC Activity of CHO expressed and purified affinity matured anti-DLL 3 trispecific proteins

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Example 5: affinity maturation to obtain affinity improved anti-DLL 3 binders

In order to obtain a more potent anti-DLL 3 conjugate, a second round of affinity maturation was performed. Phage display libraries were generated based on DH6 (SEQ ID No. 75) and DH58 (SEQ ID No. 74) parent sequences. The sequence of the binding agent for this round of affinity maturation is provided in SEQ id nos. 368 to 442. The CDR1 sequences of the DLL3 conjugates identified in this round of affinity maturation are SEQ ID nos. 810 to 884, the CDR2 sequences of the DLL3 conjugates identified in this round of affinity maturation are SEQ ID nos. 1252 to 1326, and the CDR3 sequences of the DLL3 conjugates identified in this round of affinity maturation are SEQ ID nos. 1692 to 1768. Table 7 provides CDR variations obtained in DH6 DLL3 conjugate sequences after phage display selection.

The affinity matured anti-DLL 3 sequences as identified above were cloned into expression vectors in the form of expression constructs comprising a signal domain followed by an anti-DLL 3 sequence, followed by a GGGGSGGGS linker (SEQ ID No. 1808), followed by an anti-human albumin single domain antibody 10G (SEQ ID No. 1774), followed by a GGGGSGGGS linker (SEQ ID No. 1808), followed by an anti-human CD3 antibody 2B2 (SEQ ID No. 1793), followed by a HHHHHH tag (SEQ ID No. 1819) to produce an anti-DLL 3 trispecific construct.

The anti-DLL 3 trispecific construct containing affinity-matured anti-DLL 3 binding sequences was then transfected into EXPI293 ^TM cells. These anti-DLL 3 trispecific constructs were then engineered with a protein a binding site and the amount of anti-DLL 3 trispecific construct in conditioned medium from transfected EXPI293 ^TM cells was quantified using an Octet instrument with a protein a tip. A control trispecific protein of similar molecular weight to the anti-DLL 3 trispecific protein was used as a standard.

The relative binding affinity of the anti-DLL 3 trispecific protein to human DLL3 protein was measured using a method of expressing the biotinylated version of the human DLL3 protein as a human IgG1 fusion protein using conditioned medium with known concentrations of the anti-DLL 3 trispecific protein, and the binding affinity measurement was performed in an Octet instrument with streptavidin tips. K _D measurements were performed using a single 50nM concentration of anti-DLL 3 trispecific protein, thereby achieving potency sequencing. The affinities measured are listed in table 8. All sequences tested were found to bind human DLL3, with K _D values ranging from 0.3nM to 34nM.

Conditioned medium was also tested in a T cell dependent cytotoxicity assay (see Nazarian AA, archibeque IL, nguyen YH, wang P, SINCLAIR AM, POWERSDA.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-DLL 3 trispecific protein. It is hypothesized that if the anti-DLL 3 trispecific protein directs the T-cells to kill DLL3 expressing DMS-153 cells, the viability of the DMS-153 cells should be reduced as determined by conducting the luciferase assay 48 hours after the start of the experiment. FIG. 16 shows a graph of representative TDCC data for anti-DLL 3 trispecific proteins ：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); containing the following DLL3 binding domains and for purified DH43 protein (SEQ ID No. 68) and purified DH6 protein (SEQ ID No. 75). EC ₅₀ values determined at TDCC are listed in table 9. Values range from 4.2pM to 1.5nM. The negative control measured TDCC was a GFP-targeting trispecific protein (as shown in figure 16) that did not direct T cells to kill DMS-153 cells.

Table 7: variants of CDR sequences according to the amino acid positions of DH6 and derivatives thereof

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Table 8: binding constants to human DLL3 determined using a single concentration of anti-DLL 3 trispecific protein

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Table 9: DMS-153TDCC values for affinity matured anti-DLL 3 trispecific proteins in conditioned medium using triplicates of human T cells

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Example 6: affinity maturation to obtain affinity improved anti-DLL 3 binders

Some anti-DLL 3 trispecific proteins containing DLL-3 binding sequences with the most potent TDCC activity in the assay described in example 5 and anti-DLL 3 trispecific proteins containing parent DLL3 binder DH6 were subcloned into CHO cell expression vectors and stably transfected in CHO cells (see Running Deer and Allison 2004.Biotechnol. Prog. 20:880-889). DLL3 binding sequences ：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 IDNo.430);52C4(SEQ ID No.431); and 52D4 (SEQ ID No. 432). The trispecific proteins were purified from the stable clone pool (pool) into conditioned medium using protein a and ion exchange chromatography. SDS-PAGE images of the purified proteins are provided in FIG. 17.

Affinity measurements for human and cynomolgus monkey DLL3 were performed using biotinylated DLL 3-targeted trispecific proteins immobilized on the Octet streptavidin tips at concentrations of 60nM, 20nM, 6.67nM, and 2.22 nM. The affinities determined from the measurements are listed in table 10. In this experiment, the anti-DLL 3 trispecific protein containing DH6 (parental DLL3 conjugate sequence of affinity-matured DLL3 conjugate sequence) had a K _D value of 13.5nM for human DLL3 and a K _D value of 11nM for cynomolgus monkey DLL 3. In contrast, the ten anti-DLL 3 trispecific proteins containing affinity-matured DLL3 conjugate molecules tested in this experiment ranged from 0.9 to 2.2nM for the K _D values of human DLL3 and from 1.4 to 3.4nM for the K _D values of cynomolgus monkey DLL 3. Thus, the range of affinity improvement for human DLL3 is 6.1 to 15 fold and the range of affinity improvement for cynomolgus DLL3 is 3.2 to 7.9 fold.

The purified protein was tested in TDCC assay using the same procedure as described in example 2, except that two additional DLL3 expressing cell lines, DMS-53 and NCI-H510A, were included in the assay. EC ₅₀ values from these TDCC determinations are listed in table 11, and graphs of DMS-53 and DMS-153TDCC data are provided in figures 18-19, respectively. The GFP-targeting trispecific molecules were not active in these assays (as shown in figures 18-19). The EC ₅₀ value in DMS-153 cells was improved by 2.3 to 12.1 fold, the EC ₅₀ value in NCI-H510A cells was improved by 4.5 to 31.5 fold, and the EC ₅₀ value in DMS-153 cells was improved by 8.1 to 26.1 fold compared to the parent molecule DH 6.

Table 10: affinity of purified CHO-expressed affinity-matured anti-DLL 3 trispecific proteins to human and cynomolgus monkey DLL3 proteins in vitro

Name of the name	huDLL3 K_D(nM)	cyDLL3 K_D(nM)
			DH6	13.5	11.0
51A2	1.2	2.0
			51A5	1.2	1.6
51F3	1.4	2.0
			51G2	2.0	3.4
51G10	0.9	1.4
			51H5	0.9	1.6
51X5	1.0	1.5
			52B1	1.1	1.9
52C4	2.2	3.0
			52D4	0.9	1.7

Table 11: TDCC Activity of affinity matured anti-DLL 3 trispecific proteins expressed by purified CHO in the case of DMS153, NCI-H510A and DMS53 cell lines and human T cells

Example 7: t cell dependent cytotoxicity assays using exemplary DLL3 targeted trispecific proteins comprising the DLL3 binding proteins of the present disclosure

Several exemplary DLL3 trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure (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 fig. 22-24. The trispecific proteins contain a DLL3 binding domain, an albumin binding domain (anti-ALB) and a CD3 binding domain (anti-CD 3) in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration as shown in figure 20 or in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration as shown in figure 21. TDCC the assay was performed in the presence or absence of 15mg/ml Human Serum Albumin (HSA). In this assay, luciferase-labeled NCI-H2171 (fig. 22), DMS-79 (fig. 23), SHP77 (fig. 24) or WM2664 (fig. 25) cells were combined with purified human T cells and a titration of exemplary DLL3 binding trispecific proteins in the presence or absence of albumin. It is hypothesized that if DLL3 binds to a trispecific protein directing T cells to kill DLL3 expressing NCI-H2171, DMS-79, SHP77 or WM2664 cells, then the viability of those cells should be reduced as determined by conducting a luciferase assay 48 hours after the start of the experiment. Figure 22 shows a graph of representative TDCC data for NCI-H2171 cells using a DLL3 binding trispecific protein containing the following DLL3 binding domains in TAC or CAT configuration. Figure 23 shows a graph of representative TDCC data using DMS-79 cells for DLL3 binding trispecific proteins containing the following DLL3 binding domains in either the TAC or CAT configuration. Fig. 24 shows a graph of representative TDCC data using SHP77 cells for DLL3 binding trispecific proteins containing the following DLL3 binding domains in TAC or CAT configuration. Fig. 25 shows a graph of representative TDCC data using WM2664 cells for DLL3 binding trispecific proteins containing the following DLL3 binding domains in TAC or CAT configuration. EC ₅₀ values determined at TDCC are listed in table 12. As shown and indicated by EC ₅₀ values, DLL3 with CAT orientation bound to a trispecific protein in the presence of Human Serum Albumin (HSA) (fig. 21) more effectively than DLL3 with TAC configuration bound to a trispecific protein in the TDCC assay.

Table 12: TDCC Activity of exemplary anti-DLL 3 trispecific proteins in the case of NCI-H2171, DMS-79, SHP77 and cell lines and human T cells

Example 8: exemplary DLL3 targeting binding of trispecific proteins to human T cells

In cell binding studies, human T cells were incubated with or without the presence of the exemplary DLL 3-targeted trispecific proteins (either in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration (SEQ ID No. 1891) or anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration (SEQ ID No. 1890)). Human T cells were further incubated with a secondary antibody (anti-trispecific antibody) conjugated to Alexa Fluor 647, which was able to recognize the anti-albumin domain in the exemplary trispecific molecule. Binding of the anti-trispecific antibodies was measured by flow cytometry. Robust binding of anti-trispecific antibodies was seen in the presence of exemplary DLL3 trispecific proteins in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration (right edge of graph in fig. 26) compared to cells incubated with the secondary antibody alone or cells not incubated with the exemplary trispecific protein or secondary antibody (left edge of graph in fig. 26). In the presence of the exemplary DLL3 trispecific protein in the anti-CD 3: anti-ALB: anti-DLL 3 (CAT) configuration, in addition (right edge of graph in fig. 27), robust binding of the anti-trispecific antibody was also seen compared to cells incubated with the secondary antibody alone or cells not incubated with the exemplary trispecific protein or the secondary antibody (left edge of graph in fig. 27).

Example 9: exemplary DLL3 targeting binding of 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 exemplary DLL3 targeting trispecific molecules (CAT or TAC configurations; SEQ ID nos. 1890 and 1891) or GFP targeting control trispecific molecules. After incubation, the cells were washed to remove unbound trispecific molecules and further incubated with a secondary antibody conjugated to Alexa Fluor 647 or FITC, which is capable of recognizing the anti-albumin domain in the trispecific molecule. Exemplary DLL 3-targeted trispecific molecules or control trispecific molecule binding to cells were measured by flow cytometry. Robust binding of DLL 3-targeted trispecific molecules (TAC configuration) to each cell line was observed (right peak of graph in fig. 28) compared to cells incubated with control trispecific molecules targeting GFP (left peak of graph in fig. 28). Robust binding of DLL 3-targeted trispecific molecules (CAT configuration) to each cell line was also observed (right peak of graph in fig. 29) compared to cells incubated with GFP-targeted control trispecific molecules (left peak of graph in fig. 29). In control experiments with cell line HCTI (colon cancer cell line) and NCI-H292 (lung cancer cell line) lacking DLL3 expression, similar amounts of anti-trispecific antibodies bound to cells incubated with exemplary DLL3 targeted trispecific proteins or GFP targeted control trispecific molecules (data not shown), indicating that exemplary DLL3 targeted trispecific molecules did not bind to cells lacking DLL3 expression.

Example 10: exemplary DLL3 targeting trispecific proteins directs T cell mediated killing of DLL3 expressing cancer cell lines

The purpose of this study was to assess whether exemplary DLL 3-targeted trispecific molecules were able to direct T-cells to kill DLL 3-expressing cell lines NCI-H82, SHP77, DMS53, and NCI-H2171. DLL3 expressing cells used in this study were engineered to express luciferase.

For the TDCC assay (T cell dependent cytotoxicity assay), T cells from four healthy donors (donor 2; donor 47; donor 81; donor 86) were mixed with DLL3 expressing cells and varying amounts of the exemplary DLL3 targeting trispecific proteins (CAT or TAC configuration; SEQ ID nos. 1890 and 1891) were added to the mixture. The mixture was incubated at 37℃for 48 hours. As a control, parallel experiments were performed using control trispecific molecules targeting GFP. After 48 hours, the remaining living DLL3 expressing cells were quantified using a luminescence assay. It was observed that DLL3 targeting trispecific molecules (TAC and CAT configurations) were able to effectively direct 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 TAC configuration; figures 34, 35, 36 and 37 for results using CAT configuration), whereas control GFP TRITAC molecules were unable to do so (also shown in figures 30-37). EC ₅₀ values are listed in tables 13 and 14. In addition, TDCC assays were performed with DLL3 targeted TriTAC and cell lines NCI-H292 and HCT116 that lack DLL3 expression. DLL3 targeting TriTAC was observed to be unable to direct T cells to kill these two cell lines lacking DLL3 expression (data not shown).

Table 13: EC ₅₀ values determined using TDCC for exemplary DLL 3-targeted trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in the anti-DLL 3: anti-ALB: anti-CD 3 (TAC) configuration, which were tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Table 14: EC50 values determined using TDCC for exemplary DLL 3-targeted trispecific proteins containing DLL3 binding domain 52D04 of the present disclosure in the anti-CD 3-anti-ALB-anti-DLL 3 (CAT) configuration, which were tested in the presence of Human Serum Albumin (HSA), using T cells from four different donors.

Example 11: exemplary DLL 3-targeting DLL 3-dependent activation of a trispecific protein on T cells

In this assay, T cells and NCI-H82 or DMS53 cells from 4 different healthy donors (donor 2; donor 35; donor 47; and donor 86) were incubated with exemplary DLL 3-targeted trispecific proteins (CAT or TAC configuration; SEQ ID No.1890 and SEQ ID No. 1891) for 48 hours at 37 ℃. T cells from the same donor were incubated with GFP-targeting control trispecific molecules GFP TRITAC, NCI-H82 or DMS53 cells for 48 hours at 37 ℃. After 48 hours, T cells were collected and CD69 and CD25 expression on T cells was measured by flow cytometry. In the presence of NCI-H82 or SHP77 cells and DLL3 targeted trispecific molecules, increased expression of CD69 or CD25 was detected on T cells from all 4 healthy donors, but in the absence of negative control GFP TRITAC, no increase in expression was detected, as shown in figures 38-45. Parallel experiments were performed with HCT116 cells lacking DLL3 expression. No increase in CD69 or CD25 expression was observed with DLL3 trispecific molecules tested using HCT116 cells (data not shown).

Example 12: DLL 3-dependent cytokine production by exemplary DLL 3-targeted trispecific protein-induced T cells

In this assay, T cells from healthy donors and NCI-H82 or SHP77 cells were incubated with exemplary DLL3 targeting trispecific molecules (CAT or TAC configuration; SEQ ID No.1890 and SEQ ID No. 1891) for 48 hours at 37 ℃. T cells from the same donor were incubated with GFP-targeting control trispecific molecules GFP TRITAC, NCI-H82 or DMS53 cells for 48 hours at 37 ℃. After 48 hours, conditioned medium was collected and the amount of various cytokines present in the conditioned medium was measured using an electrochemiluminescence assay (Meso Scale Discovery). It was observed that ifnγ, IL-2 and tnfα were secreted into the medium in the presence of NCI-H82 or SHP77 cells and targeting DLL3 trispecific molecules, but they were not secreted into the medium in the absence of control GFP targeting TriTAC molecules. DLL3 targeting trispecific molecules for TAC configuration: ifnγ production is shown in figures 46 and 47; IL-2 production is shown in FIGS. 48 and 49; tnfα production is shown in fig. 50 and 51. DLL3 targeting trispecific molecules for CAT configuration: ifnγ production is shown in figures 52 and 53; IL-2 production is shown in FIGS. 54 and 55; tnfα production is shown in fig. 56 and 57.

Example 13: exemplary DLL3 targeted trispecific protein inhibition of NCI-H82 xenograft growth

For this study, 5x 10 ⁶ human T cells and 5x 10 ⁶ NCI-H82 small cell lung cancer cells were injected into mice on day 0. Exemplary DLL 3-targeted trispecific molecules (CAT or TAC configurations; SEQ ID No.1890 and SEQ ID No. 1891) or negative control GFP-targeted TriTAC at a dose of 500 μg/kg were injected intraperitoneally (i.p.) into mice at a dose of 20, 100, or 500 μg/kg per day on days 1 through 10. Tumor volumes were measured every few days starting on day 7 and ending on day 24. Significant tumor growth inhibition was observed in mice injected with DLL3 targeted trispecific protein at all doses compared to mice dosed with GFP targeted TriTAC at 500 μg/kg, as shown in figure 58.

Example 14: exemplary DLL3 targeted trispecific protein elimination of NCI-H82 xenografts

For this study, 5X 10 ⁶ NCI-H82 small cell lung cancer cells were injected subcutaneously on day 0. Mice were randomized on day 8 and each mouse was injected with 2x 10 ⁷ human T cells. Exemplary DLL 3-targeted trispecific molecules (CAT configuration; SEQ ID No. 1890) at doses of 1, 10 or 100 μg/kg or negative control GFP-targeted TriTAC at doses of 100 μg/kg were injected i.p. into mice daily on days 9 through 18. Tumor volumes were measured every few days starting on day 8 and ending on day 29. Significant tumor growth inhibition was observed in mice injected with DLL3 targeted trispecific molecules at doses of 10 and 100 μg/kg compared to mice dosed with GFP targeted TriTAC at 100 μg/kg, as shown in figure 59.

Example 15: inhibition of SHP77 xenograft growth by exemplary DLL3 targeted trispecific proteins

For this study, 5x 10 ⁶ human T cells and 1x 10 ⁷ SHP77 small cell lung cancer cells were injected into mice on day 0. Mice were injected i.p. daily with either 1, 10 or 100 μg/kg of DLL3 targeted trispecific molecules (CAT configuration; SEQ ID No. 1890) or 100 μg/kg of negative control GFP targeted TriTAC on days 1 to 10. Tumor volumes were measured every few days starting on day 6 and ending on day 28. Significant tumor growth inhibition was observed in mice injected with DLL3 targeted trispecific molecules at doses of 10 and 100 μg/kg compared to mice dosed with GFP targeted TriTAC at 100 μg/kg, as shown in figure 60.

Example 16: exemplary DLL3 pharmacokinetic profile of trispecific proteins

When administered at 0.3mg/kg, the half-life of the DLL3 targeted trispecific protein in cynomolgus monkeys is about 3 to about 3.9 days

For this study, cynomolgus monkeys were intravenously injected with a dose of 0.3mg/kg of an exemplary DLL 3-targeted trispecific molecule (CAT or TAC configuration; SEQ ID No.1890 and SEQ ID No. 1891), and serum samples were collected at different time points after injection. Two monkeys were injected for each dose. In electrochemiluminescence assays, the amount of DLL3 targeted trispecific molecules in serum was measured using an anti-idiotype antibody that recognizes the trispecific molecule. Figure 61 shows graphs of serum DLL3 targeted trispecific molecular levels at different time points. The data were then used to calculate the pharmacokinetic properties of DLL3 targeted trispecific molecules as provided in table 15. Based on pharmacokinetic data, a human dosing schedule of once or twice a week is envisaged.

Table 15: exemplary DLL3 pharmacokinetics of the targeted trispecific molecules

When administered at 1 or 10mg/kg, the half-life of the DLL3 targeted trispecific protein in cynomolgus monkeys is about 2.8 to about 3.3 days:

For this study, cynomolgus monkeys were intravenously injected with 1mg/kg or 10mg/kg doses of exemplary DLL3 targeted trispecific molecules, and serum samples were collected at different time points after injection. Two monkeys were injected for each dose. In an electrochemiluminescence assay, the amount of DLL3 targeted TriTAC in serum was measured using an anti-idiotype antibody that recognizes TriTAC molecules. Figure 62 shows graphs of serum DLL3 targeted trispecific molecular levels at different time points. The pharmacokinetic properties of TriTAC molecules were then calculated using the data, as provided in table 16. Pharmacokinetic data indicate that administration is once or twice a week in humans.

Table 16: exemplary DLL3 pharmacokinetics of the targeted trispecific molecules

Cynomolgus monkey tolerance exemplary DLL3 targets a trispecific protein when administered in a single dose up to 10 mg/kg:

A transient increase in serum cytokine levels was observed, mainly upon administration of the exemplary DLL3 targeted trispecific protein (CAT configuration) at a dose of 10mg/kg (FIG. 63; IFNγ, upper panel of FIG. 63, IL-6, second panel of FIG. 63; IL-10, third panel of FIG. 63). Transient T cell edge sets and T cell activation were also observed (data not shown). At end and convalescent periods euthanasia, no macroscopic findings or organ weight differences were observed with DLL3 trispecific proteins, and at convalescent periods euthanasia, no microscopic findings with DLL3 trispecific proteins were observed.

To demonstrate that DLL3 targeting TriTAC retains cell-directed killing activity after administration to cynomolgus monkeys, serum samples from the 10mg/kg dose group collected 168h post-dose were tested in the DMS53 TDCC assay and compared to freshly thawed DLL3 targeting trispecific TriTAC. The same cell DMS53 cell killing was observed using serum samples and freshly thawed proteins (fig. 64), indicating that DLL3 targeting TriTAC retains the ability to direct T cell killing of target cells 1 week after administration to cynomolgus monkeys.

Example 17: xenograft tumor model

Exemplary anti-DLL 3 targeted trispecific proteins of the present disclosure were evaluated in xenograft models.

Female immunodeficient NOD/scid mice were sub-lethally irradiated (2 Gy) and 1x 10 ⁶ NCI-H28 cells were inoculated subcutaneously into their right dorsal side. When the tumors reached 100 to 200mm ³, the animals were divided into 3 treatment groups. Groups 2 and 3 (8 animals per group) were injected intraperitoneally with 1.5x10 ⁷ activated human T cells. Three days later, animals of group 3 (qdx d) were then treated with a total of 9 intravenous doses of the exemplary DLL3 trispecific antigen-binding protein, such as 1, 10, 50, or 100 μg/kg. Groups 1 and 2 were treated with vehicle only. Body weight and tumor volume were determined for 30 days.

Animals treated with the exemplary DLL3 targeted trispecific proteins of the previous examples were expected to have a statistically significant delay in tumor growth compared to the corresponding vehicle treated control group.

Example 18: proof of concept clinical trial protocol for administration of exemplary DLL3 trispecific antigen-binding proteins (anti-DLL 3 trispecific proteins) to neuroendocrine cancer patients

This is a phase I/II clinical trial for studying exemplary DLL3 trispecific antigen-binding proteins as a treatment for neuroendocrine cancer.

Study results:

mainly: exemplary DLL3 targeted to the maximum tolerated dose of the trispecific protein

Secondary: determining whether the in vitro response of the exemplary DLL3 targeted trispecific protein correlates with a clinical response

Stage I

The Maximum Tolerated Dose (MTD) will be determined during the phase I portion of the trial.

1.1 The Maximum Tolerated Dose (MTD) will be determined in the phase I portion of the trial.

1.2 Patients meeting the qualification criteria will enter the trial to evaluate exemplary DLL3 targeted trispecific proteins.

1.3 The goal was to identify the highest dose at which the exemplary anti-DLL 3 trispecific proteins could be safely administered without producing serious or unmanageable side effects in the participants. The dose administered will depend on the number of participants recruited in the previous study and the dose tolerance. Not all participants will receive the same dose.

Phase II

2.1 The subsequent phase II portion will be treated with MTD with the objective of determining whether therapy with the exemplary DLL3 targeted trispecific protein therapy results in a response rate of at least 20%.

Major outcome of phase ii— determining whether therapy targeting the trispecific protein with exemplary DLL3 achieves clinical response (blast response), mild response, partial response, or complete response) in at least 20% of patients.

Qualification of: biopsies demonstrated neuroendocrine tumors that were positive for somatostatin receptors, as demonstrated for somatostatin receptor PET.

All sites or origins are acceptable.

Both functional and non-functional tumors are permissible.

The tumor should not be reduced by operation.

ECOG behavior states 0,1 or 2

Age >18 years.

Can understand written informed consent and is willing to sign.

Example 19: DLL3 trispecific antigen binding proteins 1/2a phase dose escalation, expansion, safety and pharmacokinetic studies

Target population: patients with Small Cell Lung Cancer (SCLC) that recurs after platinum-based chemotherapy, or patients with standard of care (SOC) recurrences/refractory (R/R) or other malignancies with high-grade neuroendocrine characteristics, including neuroendocrine prostate cancer (NEPC) and other neuroendocrine neoplasms (NEN), for which no SOC is available.

Test purpose: safety and tolerability at increasing dose levels were assessed, PK and pharmacodynamic data were determined and primary anti-tumor activity was assessed.

And (3) test design: the DLL3 trispecific antigen-binding protein phase 1/2 assay design is shown in figure 65. The test objective was to evaluate safety and tolerability at increasing dose levels, determine PK and pharmacodynamic data and evaluate preliminary antitumor activity

Dosing and administration: DLL3 trispecific antigen-binding protein (SEQ ID NO: 1890) was administered once weekly by infusion starting at 15 μg corresponding to EC50 (fixed dose). One cycle was 21 days with three doses. The patient received preoperative medications with dexamethasone, tenol (tylnol) and histamine receptor blockers at an initial dose. Table 17 shows the dosing cohort and the number of subjects. Once weekly administration is tolerated and no Dose Limiting Toxicity (DLT) has been observed to date. Table 18 shows baseline demographics for these patients. The median number of previous systemic therapies was 2 and ranged from 1-5. 77.8% of patients were previously exposed to immune checkpoint inhibitors, including 100% of SCLC patients.

Table 17: DLL3 trispecific antigen-binding protein administration queues

Table 18: patient baseline demographics

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* Other NEN: retroperitoneal (primary focus unknown), colon, pancreas, thymus, bladder

Treatment time: the median treatment duration was 11.6 weeks, ranging from 4.1 to 41.4 weeks. 6 of the 10 patients (33%) received treatment for more than 20 weeks. Figure 66 shows the treatment time, weekly dose, and number of previous treatments for the patient.

Safety and tolerability: dose Limiting Toxicity (DLT) was not observed. There were [4 (22%) ] patients reporting class 1-2 CRS and no class 3 CRS. Immune effector cell-associated neurotoxicity syndrome was not reported (ICANS). No patient interruption due to adverse events.

Table 19. Treatment by grade highlights adverse events (TEAE) ^a

^a Classification according to CTCAE v1.0 except for cytokine release syndrome (classification according to ASTCT 2019)

Target lesion response: there was any reduction in the sum of the diameters of 7 (38.9%) target lesions in 18 patients, including 5 SCLCs, 1 NEPC and 1 NEN (thymic atypical carcinoid). 1 SCLC 2L patient had confirmed a partial response and was receiving treatment for 32 weeks. For SCLC patients, the sum of target lesion diameters for 3 out of 11 patients (27.3%) at all doses was reduced by >30%. 6 of the 18 patients (33%) showed the best overall response with disease stability, including 1 SCLC, 1 NEPC and 1 NEN.

Patient 102 profile: patient 102 was a 71 year old female diagnosed with SCLC at 9 months 2020. Treatment was initiated at 45ng/kg and showed a 38% decrease at week 9, which was an unproven Partial Response (PR) (fig. 68). To date, no adverse treatment-related effects (AEs) were observed for patient 102 and were still under study after 9 weeks of treatment.

Table 20: patient 102 baseline characteristics

Fig. 69 shows pharmacokinetic data for DLL3 trispecific antigen-binding proteins from different drug administration queues. An increase in half-life of about 70 hours was observed, and serum Cmax increased with increasing dose.

Fig. 70 shows the results of the flow analysis. Figure 70A shows T cell edge levels after treatment. The presence of dose-dependent and transient peripheral T cell edge sets was shown. Figure 70B shows induction of activation markers after treatment. T cell activation was observed in the 135 μg/week cohort, which supports T cell activation in vivo.

Patient 111 profile: patient 111 was a61 year old female diagnosed with extensive SCLC at month 1 of 2021. The selected Target Lesions (TL) metastasize to one place in the lung, two places in the liver and two places in the lymph nodes. Non-target lesions (not TL) metastasize to two sites in the lung and two sites in the liver. Previous systemic treatments included carboplatin etoposide and atilizumab for 20.1 weeks. After entering the study, the disease stabilized to the best response to the last previous systemic treatment. Treatment was initiated at 1215 μg/week and the dose was increased to 3600 μg/week starting from C3D15 (week 8), followed by an increase in dose to 7000 μg/week. Week 10 confirmed Partial Response (PR), the sum of target lesion diameters decreased by 53.3%, and the patient continued treatment for more than 32 weeks.

Table 21: patient 111 baseline characteristics

Fig. 71A shows the change in target lesions of patient 111 over time. The CT scan of FIG. 71B shows a reduction in the sum of the diameters of the target lesions of patient 111. Target lesion diameter was reduced by 38.1% at week 6 after treatment and 53.3% at week 10 after treatment.

Patient 112 profile: patient 112 was a 67 year old female diagnosed with extensive SCLC at month 4 of 2020. TL metastasizes to two sites in the liver and two sites in the lymph nodes. non-TL are in liver, lymph nodes, spleen, bone and brain. Previous systemic treatments included carboplatin, etoposide and terlipressin Li Shan (anti-PD 1), 2 cycles of cisplatin and etoposide, and lubiperidine (Lurbinectedin) for 4 cycles in clinical trials. The time of the last previous systemic treatment was 10.9 weeks. After entering the study, the partial response was the best response to the last previous systemic treatment. Patient 112 received a step dose (3.600 μg/week followed by 7,200 μg/week) treatment. At week 9, a 27% decrease in the sum of the diameters of the target lesions was observed, mainly in lymph nodes, and liver metastasis was stable, symptoms improved, and patients continued treatment for more than 10 weeks. At week 27, a 64.6% decrease in the sum of the target lesion diameters from baseline was observed, and patient 112 continued treatment for more than 28 weeks.

Table 22: patient 112 baseline characteristics

Fig. 72A shows the change in target lesions of patient 112 over time. The CT scan of FIG. 72B shows a reduction in the sum of the diameters of the target lesions of patient 112.

Patient 113 profile: patient 113 was a 65 year old male diagnosed with neuroendocrine prostate cancer at 11 months 2020. TL metastasize to two sites in the lung, one site in the liver and two sites in the lymph nodes. non-TL are in the lung, liver, lymph nodes and prostate. Previous systemic treatments included cisplatin and etoposide, and CAV. The last previous systemic treatment was 4 weeks. After entering the study, the disease progressed to the optimal response to the last previous systemic treatment. Patient 113 received a step dose (3600 μg/week followed by 7200 μg/week) treatment. At week 9, a 15.3% decrease in the sum of the diameters of the target lesions, a reduction in lung lesions and prostate, a new lesion identified in the liver, an improvement in quality of life, a significant reduction in urinary symptoms and pain was observed, and the patient was still under study for more than 10 weeks. Fig. 73 shows the change in target lesions of patient 113 over time.

The DLL3 trispecific antigen binding protein used in this study showed linear PK, an increase in exposure dose ratio from 0.135mg to 12mg, and a median half-life of 71 hours.

T cell edge sets were observed and consistent with target binding. A small transient increase in serum IL-6 and MCP-1 was observed up to 24 hours post-administration. A "first dose" effect was observed, with smaller edge sets and lower median IL-6 and MCP-1 concentrations in the case of repeated or target doses.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims are intended to define the scope of the invention and their equivalents and methods and structures within the scope of these claims are hereby contemplated.

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CD3 binding domain CDR sequences

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DLL3 protein UniProtKB accession number Q9NYJ7 (SEQ ID NO: 1885)

> Sp|q9nyj7|dll3_human delta-like protein 3os=homo sapiens ox=9606 gn=dll3pe=1sv=1

MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFF

RVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTF

SFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEP

PAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCL

EGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTC

PRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQ

PCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALG

FGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAP

PGLRPGDPQRYLLPPALGLLVAAGVAGAALLLVHVRRRGHSQDAGSRLLAGTPEPSVHAL

PDALNNLRTQEGSGDGPSSSVDWNRPEDVDPQGIYVISAPSIYAREVATPLFPPLHTGRA

GQRQHLLFPYPSSILSVK

51X5(SEQ ID NO: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 [ Chile ] (SEQ ID No. 1892)

MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSE

EAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAW

SLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPL

EDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDG

NPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNC

EKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALG

FGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQR

YLLPPALGLLVAAGVAGAALLLVHVRRRGHSQDAGSRLLAGTPEPSVHALPDALNNLRTQEGSGDGPSSS

VDWNRPEDVDPQGIYVISAPSIYAREVATPLFPPLHTGRAGQRQHLLFPYPSSILSVK

DLL3 protein sequence (SEQ ID NO: 1893)

RSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYL

Claims

1. A method of treating cancer, the method comprising administering to a subject an effective amount of a delta-like ligand 3 (DLL 3) targeted trispecific protein, wherein the protein comprises

(A) A first domain (a) that specifically binds human CD3;

(b) A second domain (B) which is a half-life extending domain; and

(C) A third domain (C) which specifically binds DLL3,

Wherein the DLL3 targeted trispecific protein is administered at a dose of about 1 μg to about 100 mg.

2. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 1 μg to about 14 mg.

3. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 1 μg to about 5 mg.

4. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 1 μg to about 2 mg.

5. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 1 μg to about 1 mg.

6. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 15 μg to about 3600 μg.

7. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 15 μg.

8. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 45 μg.

9. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 135 μg.

10. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 405 μg.

11. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 1215 μg.

12. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 3600 μg.

13. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 5 mg.

14. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 7 mg.

15. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 10 mg.

16. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 12 mg.

17. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 14 mg.

18. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 20 mg.

19. The method of claim 1 wherein the DLL3 targeted trispecific protein is administered at a dose of about 50 mg.

20. The method of any one of claims 1-19, wherein the DLL3 targeted trispecific protein is administered once per week.

21. The method of any one of claims 1-19 wherein the DLL3 targeted trispecific protein is administered twice per week.

22. The method of any one of claims 1-19 wherein the DLL3 targeted trispecific protein is administered every other week.

23. The method of any one of claims 1-19 wherein the DLL3 targeted trispecific protein is administered every three weeks.

24. The method of any one of claims 1-23, wherein the DLL3 targets the tri-specific protein for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

25. A method of treating cancer, the method comprising administering to a subject an effective amount of a DLL3 targeted trispecific protein, wherein the protein comprises

(A) A first domain (a) that specifically binds human CD3;

(b) A second domain (B) which is a half-life extending domain; and

(C) A third domain (C) which specifically binds DLL3,

Wherein the domains are linked in the order H ₂ N- (a) - (B) - (C) -COOH, or through linkers L1 and L2, and wherein the DLL 3-targeted trispecific protein is administered according to a schedule comprising the steps of:

(i) Administering a first dose of the DLL3 targeted trispecific protein, and

(Ii) Administering a second dose of the DLL3 targeted trispecific protein, wherein the second dose is higher than the first dose.

26. The method of claim 25, wherein the first dose is about 1mg to about 50mg.

27. The method of claim 25, wherein the first dose is about 1mg to about 20mg.

28. The method of claim 25, wherein the first dose is about 1mg to about 10mg.

29. The method of claim 25, wherein the first dose is about 1mg to about 5mg.

30. The method of claim 25, wherein the first dose is about 1mg to about 3mg.

31. The method of claim 25, wherein the first dose is about 2000 μg.

32. The method of claim 25, wherein the first dose is about 3600 μg.

33. The method of any one of claims 25-32, wherein the first dose is administered for about 1 week to about 36 weeks.

34. The method of any one of claims 25-32, wherein the first dose is administered for about 1 week to about 27 weeks.

35. The method of any one of claims 25-32, wherein the first dose is administered for about 1 week to about 18 weeks.

36. The method of any one of claims 25-32, wherein the first dose is administered for about 1 week to about 9 weeks.

37. The method of any one of claims 25-36, wherein the first dose is administered once daily.

38. The method of any one of claims 25-36, wherein the first dose is administered twice daily.

39. The method of any one of claims 25-36, wherein the first dose is administered three times per day.

40. The method of any one of claims 25-36, wherein the first dose is administered five times per day.

41. The method of any one of claims 25-36, wherein the first dose is administered once a week.

42. The method of any one of claims 25-36, wherein the first dose is administered twice a week.

43. The method of any one of claims 25-36, wherein the first dose is administered every other week.

44. The method of any one of claims 25-36, wherein the first dose is administered every three weeks.

45. The method of any one of claims 25-44, wherein the first dose is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally.

46. The method of claim 25, wherein the second dose is about 1mg to about 100mg.

47. The method of claim 25, wherein the second dose is about 1mg to about 50mg.

48. The method of claim 25, wherein the second dose is about 50mg to about 100mg.

49. The method of claim 25, wherein the second dose is about 7.2mg.

50. The method of claim 25, wherein the second dose is about 12mg.

51. The method of claim 25, wherein the second dose is about 24mg.

52. The method of any one of claims 25-51, wherein the second dose is administered for about 1 week to about 36 weeks.

53. The method of any one of claims 25-51, wherein the second dose is administered for about 1 week to about 27 weeks.

54. The method of any one of claims 25-51, wherein the second dose is administered for about 1 week to about 18 weeks.

55. The method of any one of claims 25-51, wherein the second dose is administered for about 1 week to about 9 weeks.

56. The method of any one of claims 25-55, wherein the second dose is administered once daily.

57. The method of any one of claims 25-55, wherein the second dose is administered twice daily.

58. The method of any one of claims 25-55, wherein the second dose is administered three times per day.

59. The method of any one of claims 25-55, wherein the second dose is administered five times per day.

60. The method of any one of claims 25-55, wherein the second dose is administered once a week.

61. The method of any one of claims 25-55, wherein the second dose is administered twice weekly.

62. The method of any one of claims 25-55, wherein the second dose is administered every other week.

63. The method of any one of claims 25-55, wherein the second dose is administered every three weeks.

64. The method of any one of claims 25-63, wherein after administration of the first dose, the second dose is maintained until the end of the plan.

65. The method of any one of claims 25-64, wherein the second dose is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally.

66. The method of any one of claims 1-65, wherein the DLL3 targeted 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.

67. The method of any one of claims 1-66, wherein the third domain comprises a VHH domain.

68. The method of claim 67, wherein the VHH domain is a human domain, a humanized domain, an affinity maturation domain, or a combination thereof.

69. The method of any one of claims 1-68, wherein the third domain comprises one or more sequences selected from SEQ ID NOs 1-442.

70. The method of any one of claims 1-69, wherein the first domain comprises a variable light chain and a variable heavy chain, each capable of specifically binding human CD3.

71. The method of claim 70, wherein the first domain is a humanized domain or a human domain.

72. The method of any one of claims 1-71, wherein the second domain binds human serum albumin.

73. The method of claim 72, wherein the second domain comprises an scFv, a variable heavy chain domain (VH), a variable light chain domain (VL), a peptide, a ligand, or a small molecule.

74. The method of any one of claims 1-73, wherein each linker L1 and L2 is independently selected from (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), wherein n is 1, 2, 3, 4, 5, 6,7, 8, 9, or 10.

75. The method of any one of claims 1-73, wherein each linker L1 and L2 is independently (GGGGS) ₄(SEQ ID NO:1817)、(GGGGS)₃ (SEQ ID NO: 1818) or GGGGSGGGS (SEQ ID NO: 1808).

76. The method of any one of claims 1-75, wherein the domains are linked in the order H ₂ N- (C) -L1- (B) -L2- (a) -COOH.

77. The method of any one of claims 1-76 wherein the DLL3 targeted trispecific protein is less than about 80kDa.

78. The method of any one of claims 1-76 wherein the DLL3 targeted trispecific protein is about 50 to about 75kDa.

79. The method of any one of claims 1-76 wherein the DLL3 targeted trispecific protein is less than about 60kDa.

80. The method of any one of claims 1-79 wherein the DLL3 targeted trispecific protein comprises a sequence selected from the group consisting of SEQ ID NO:1890-SEQ ID NO: 1891.

81. The method of any one of claims 1-79 wherein the DLL3 targeted trispecific protein comprises a sequence as set forth in SEQ ID No. 1890.

82. The method of any one of claims 1-81, wherein the cancer is a neoplastic disease, an autoimmune disease, or an infectious disease associated with DLL 3.

83. The method of any one of claims 1-81, wherein the cancer is neuroendocrine, prostate, lung, gastric, squamous cell, pancreatic, cholangiocarcinoma, triple negative breast or ovarian cancer.

84. The method of any one of claims 1-81, wherein the cancer is small cell lung cancer.

85. The method of any one of claims 1-81, wherein the cancer is neuroendocrine prostate cancer.