DLL3 binding proteins and methods of use

Abstract

SOLUTION: Provided herein are DLL3 binding proteins and DLL3 targeting multispecific proteins (e.g., DLL3 targeting trispecific protein) comprising a domain binding to CD3, a half-life extension domain, and a domain binding to DLL3 (such as a DLL3 binding protein as provided herein). Also provided are pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such DLL3 binding proteins, DLL3 targeting trispecific proteins. Also disclosed are methods of using the DLL3 binding proteins, DLL3 targeting trispecific proteins in the prevention, and / or treatment of diseases, conditions and disorders.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 736,368, filed Sep. 25, 2018; U.S. Provisional Patent Application No. 62 / 736,358, filed Sep. 25, 2018; and U.S. Provisional Patent Application No. 62 / 877,227, filed Jul. 22, 2019, the entire disclosures of which are hereby incorporated by reference in their entirety.

[0002] Sequence Listing This application includes a sequence listing which is submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on Sep. 25, 2019, is named 47517-733_601_SL.txt and is 951,519 bytes in size.

Background Art

[0003] The selective destruction of individual cells or specific cell types is often desirable in various clinical settings. For example, a major goal of cancer treatment is to specifically destroy tumor cells while leaving healthy cells and tissues intact without damage. One such method is to elicit an immune response against the tumor in order to attack and destroy the tumor cells by immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs).

Summary of the Invention

[0004] There remains a need for additional options available for the treatment of neoplastic diseases associated with overexpression of DLL3, such as neuroendocrine tumors. In certain embodiments, the present disclosure provides single domain proteins that specifically bind to DLL3 on the surface of tumor target cells and multispecific proteins, such as trispecific proteins containing a DLL3 binding domain as described herein. In some embodiments, the present disclosure provides a delta-like ligand 3 (DLL3) binding protein, or a multispecific protein as described above, which can be used to diagnose and treat symptoms that are mutually related to the expression of DLL3.

[0005] One embodiment provides a DLL3-targeted trispecific protein, wherein the DLL3-targeted trispecific protein comprises: (a) a first domain (A) that is a single-chain variable fragment that specifically binds to human CD3; (b) a second domain (B) that is a single domain antibody that specifically binds to human serum albumin protein; and (c) a third domain (C) that is a single domain antibody that specifically binds to DLL3 protein.

[0006] In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, H2N-(C)-(B)-(A)-COOH, or H2N-(C)-(A)-(B)-COOH, or by linkers L1 and L2 in the order of H2N-(A)-L1-(B)-L2-(C)-COOH, H2N-(A)-L1-(C)-L2-(B)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2N-(B)-L1-(C)-L2-(A)-COOH, H2N-(C)-L1-(B)-L2-(A)-COOH, or H2N-(C)-L1-(A)-L2-(B)-COOH.

[0007] In some embodiments, the third domain (C) comprises an affinity matured binding molecule. In some embodiments, the affinity matured binding molecule is derived from a parental molecule that specifically binds to the DLL3 protein. In some embodiments, the affinity matured DLL3-binding molecule has a binding affinity for the DLL3 protein that is about 2-fold to about 50-fold greater than the binding affinity of the parental molecule for the DLL3 protein. In some embodiments, the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 1-442 and 1886. In some embodiments, the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 1-52. In some embodiments, the third domain (C) comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID No. 443-884 and 1887, wherein CDR2 comprises a sequence selected from the group consisting of SEQ ID No. 885-1326 and 1888, and wherein CDR3 comprises a sequence selected from the group consisting of SEQ ID No. 1327-1768 and 1889. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID No. 495-528. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID No. 937-970. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID No. 1379-1412. In some embodiments, the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 53-86. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID No. 529-809. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID No. 971-1251. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID No. 1379-1412.In some embodiments, the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 87 - 367. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID No. 810 - 884. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID No. 1252 - 1326. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID No. 1692 - 1768. In some embodiments, the DLL3 - targeting trispecific protein of claim 1, wherein the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 368 - 442.

[0008] One embodiment provides a DLL3 - targeting trispecific protein, wherein the DLL3 - targeting trispecific protein comprises (a) a first domain (A) which is a single - chain variable fragment that specifically binds to human CD3, (b) a second domain (B) which is a single - domain antibody that specifically binds to human serum albumin protein, and (c) a third domain (C) which is a single - domain antibody that specifically binds to DLL3, wherein the third domain comprises the sequence of SEQ ID No. 68 or SEQ ID No. 75, or is derived from SEQ ID No. 68 or SEQ ID No. 75.

[0009] In some embodiments, the third domain (C) is derived from SEQ ID No. 75. In some embodiments, the third domain comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the following sequence: X1X2X3X4X5X6X7SX8A, CDR2 comprises the following sequence: GJ1SJ2J3GJ4J5J6YJ7J8SVKG (SEQ ID No. 1894), and CDR3 comprises the following sequence: Z1Z2Z3Z4Z5Z6Z7Z8Z9.

[0010] In some embodiments, X1 is A, D, E, F, G, H, K, L, M, N, Q, R, S, V, W, or Y, X2 is D, E, H, K, M, P, R, S, T, or Y, X3 is A, D, G, H, K, N, P, Q, R, S, T, V, or Y, X4 is K, S, or V, X5 is A, F, G, H, K, L, M, N, Q, R, S, T, V, W, or Y, X6 is D, F, H, I, K, L, M, N, Q, R, S, V, or Y, X7 is L or M, and, X8 is I, L, M, S, T, or V.

[0011] In some embodiments, J1 is I or V, J2 is A, D, E, G, H, I, K, L, N, P, Q, R, S, T, V, or Y, J3 is A, D, E, G, H, N, R, or T, J4 is H, P, R, or S, J5 is A, H, I, K, M, N, Q, R, T, or V, J6 is A, D, G, H, I, L, M, N, S, T, V, or Y, J7 is A, F, I, L, M, R, S, T, V, or Y, J8 is A, D, E, G, H, K, L, N, R, S, or V;

[0012] In some embodiments, <x Z1 is L or Y, Z2 is D, E, G, H, K, N, Q, R, S, T, V, or Y, Z3 is Q or W, Z4 is A, D, E, G, H, I, K, L, M, P, R, S, T, or V, Z5 is A, D, E, G, N, R, S, T, or Y, Z6 is A, P, R, or S, Z7 is A, D, F, G, H, L, M, N, Q, R, S, T, V, or Y, Z8 is A, G, I, K, P, Q, R, S, or T, and, Z9 is F, H, or Y.

[0013] In some embodiments, linkers L1 and L2 are each independently (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), or (GGGGS) n (SEQ ID No.1814), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or GGGGSGGGS (SEQ ID No.1808). In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH, or, by linkers L1 and L2, in the order of H2N-(A)-L1-(B)-L2-(C)-COOH.

[0014] One embodiment provides a DLL3 binding protein comprising the following formula, f1-r1-f2-r2-f3-r3-f4 Here, r1 is Complementary Determining Region 1 (CDR1) and is identical to or includes one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID No. 443-884 and 1887, r2 is CDR2 and is identical to or includes one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID No. 885-1326 and 1888, and r3 is CDR3 and is identical to or includes one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID No. 1327-1768 and 1889, where f1, f2, f3, and f4 are framework residues.

[0015] One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, the method comprising administering to a subject a DLL3-targeting trispecific protein according to the present disclosure.

[0016] One embodiment provides a DLL3-targeted trispecific protein, wherein the DLL3-targeted trispecific protein comprises: (a) a first domain (A) that specifically binds to human CD3; (b) a second domain (B) that is a half-life extension domain; and (c) a third domain (C) that specifically binds to the DLL3 protein. In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, H2N-(C)-(B)-(A)-COOH, or H2N-(C)-(A)-(B)-COOH. In some embodiments, the third domain (C) comprises an affinity matured binding molecule. In some embodiments, the affinity matured binding molecule is derived from a parental molecule that specifically binds to the DLL3 protein. In some embodiments, the affinity matured binding molecule is derived from a parental molecule that specifically binds to DLL3 after rounds of affinity maturation. In some embodiments, the rounds of affinity maturation include panning a phage display library against the DLL3 protein. In some embodiments, the phage display library is generated by mutating one or more residues of the parental molecule. In some embodiments, the phage display library expresses one or more molecules derived from the parental molecule. In some embodiments, the affinity matured binding molecule is selected from one or more molecules derived from the parental molecule. In some embodiments, the affinity matured DLL3 binding molecule has a binding affinity for the DLL3 protein that is greater than the binding affinity of the parental molecule for the DLL3 protein. In some embodiments, the affinity matured binding DLL3 molecule has a binding affinity for the DLL3 protein that is about 2-fold, about 50-fold greater than the binding affinity of the parental molecule for the DLL3 protein. In some embodiments, the affinity matured binding DLL3 molecule has a binding affinity for the DLL3 protein that is about 3-fold greater than the binding affinity of the parental molecule for the DLL3 protein. In some embodiments, the third domain (C) comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID No. 1-442 and 1886.In some embodiments, the third domain (C) comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-52. In some embodiments, the third domain (C) comprises CDR1, CDR2, and CDR3.

[0017] In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 443-884 and 1887, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 443-884 and 1887. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 885-1326 and 1888, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 885-1326 and 1888. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889, or one or more substitutions relative to a sequence selected from SEQ ID Nos. 1327-1768 and 1889. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 495-528, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 495-528. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 937-970, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 937-970. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1379-1412, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 1379-1412.

[0018] In some embodiments, the third domain (C) comprises a sequence that is at least about 80% identical to a sequence selected from the group consisting of SEQ ID No. 53 - 86. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID No. 529 - 809, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID No. 529 - 809. In some embodiments, CDR2 comprises a sequence having one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID No. 971 - 1251. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID No. 1379 - 1412, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID No. 1379 - 1412. In some embodiments, the third domain (C) comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID No. 87 - 367. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID No. 810 - 884, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID No. 810 - 884. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID No. 1252 - 1326, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID No. 1252 - 1326. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID No. 1692 - 1768, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID No. 1692 - 1768. In some embodiments, the third domain (C) comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID No. 368 - 442.

[0019] One embodiment provides a DLL3-targeted trispecific protein, wherein the DLL3-targeted trispecific protein comprises: (a) a first domain (A) that specifically binds to human CD3, (b) a second domain (B) that is a half-life extension domain, and (c) a third domain (C) that specifically binds to DLL3, wherein the third domain comprises the sequence of SEQ ID No. 68 or is derived from SEQ ID No. 68. In some embodiments, the third domain (C) is derived from SEQ ID No. 68. In some embodiments, SEQ ID No. 68 comprises CDR1, CDR2, and CDR3. In some embodiments, CDR1 comprises the following sequence: GX1X2X3X4X5NX6X7X8. In some embodiments, CDR2 comprises the following sequence: GJ1SJ2J3J4J5J6J7J8J9J 10 SJ 11 KJ 12 (SEQ ID No. 1895).

[0020] In some embodiments, CDR3 comprises the following sequence: Z1Z2Z3Z4Z5Z6Z7Z8Z9Z 10 Z 11 and. In some embodiments, X1 is A, E, F, G, I, K, L, N, Q, R, S, T, V, or Y, X2 is A, G, I, K, P, R, S, T, or V, X3 is A, D, F, K, L, N, P, Q, R, S, T, or Y, X4 is A, D, F, H, I, K, L, M, N, P, R, S, T, V, or Y, X5 is F, I, K, L, M, N, R, S, T, or V, X6 is A or G, X7 is F, I, L, M, T, V, or Y, and, X8 is A or G.

[0021] In some embodiments, J1 is I or V, J2 is A, K, P, R, or S, J3 is D or N, J4 is D, E, G, K, N, Q, R, S, T, or Y, J5 is S or T, J6 is A, E, F, H, I, K, L, N, Q, R, S, T, V, or Y, J7 is A, I, L, M, V, or Y, J8 is D, F, H, I, L, N, S, T, V, or Y, J9 is A, D, E, F, G, I, K, L, N, Q, R, S, T, V, or Y, J 10 is A, D, E, G, K, Q, S, or V, J 11 is A or V, and, J 12 is G or V.

[0022] In some embodiments, Z1 is F or Y, Z2 is G, H, I, K, N, R, S, or T, Z3 is A, F, H, I, K, L, M, N, P, Q, R, S, T, or Y, Z4 is A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, or Y, Z5 is A, C, D, E, G, H, I, K, L, N, P, Q, R, S, T, W, or Y, Z6 is G, K, L, R, or T, Z7 is A, G, H, L, Q, R, S, T, V, or Y, Z8 is A, D, E, G, H, P, Q, S, T, W, or Y, Z9 is A, G, I, K, L, M, N, Q, R, S, T, V, or Y, Z 10 is A, G, K, P, R, S, T, or V, and, Z 11 is A, F, S, or Y.

[0023] One embodiment provides a DLL3-targeting trispecific protein, wherein the DLL3-targeting trispecific protein comprises: (a) a first domain (A) that specifically binds to human CD3; (b) a second domain (B) that is a half-life extension domain; and (c) a third domain (C) that specifically binds to DLL3, wherein the third domain comprises the sequence of SEQ ID No. 75 or is derived from SEQ ID No. 75. In some embodiments, SEQ ID No. 75 comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the following sequence: X1X2X3X4X5X6X7SX8A, CDR2 comprises the following sequence: GJ1SJ2J3GJ4J5J6YJ7J8SVKG (SEQ ID No. 1894), and CDR3 comprises the following sequence: Z1Z2Z3Z4Z5Z6Z7Z8Z9.

[0024] In some embodiments, X1 is A, D, E, F, G, H, K, L, M, N, Q, R, S, V, W, or Y; X2 is D, E, H, K, M, P, R, S, T, or Y; X3 is A, D, G, H, K, N, P, Q, R, S, T, V, or Y; X4 is K, S, or V; X5 is A, F, G, H, K, L, M, N, Q, R, S, T, V, W, or Y; X6 is D, F, H, I, K, L, M, N, Q, R, S, V, or Y; X7 is L or M, and X8 is I, L, M, S, T, or V.

[0025] In some embodiments, J1 is I or V; J2 is A, D, E, G, H, I, K, L, N, P, Q, R, S, T, V, or Y; J3 is A, D, E, G, H, N, R, or T; J4 is H, P, R, or S; J5 is A, H, I, K, M, N, Q, R, T, or V; J6 is A, D, G, H, I, L, M, N, S, T, V, or Y, J7 is A, F, I, L, M, R, S, T, V, or Y, J8 is A, D, E, G, H, K, L, N, R, S, or V;

[0026] In some embodiments, Z1 is L or Y, Z2 is D, E, G, H, K, N, Q, R, S, T, V, or Y, Z3 is Q or W, Z4 is A, D, E, G, H, I, K, L, M, P, R, S, T, or V, Z5 is A, D, E, G, N, R, S, T, or Y, Z6 is A, P, R, or S, Z7 is A, D, F, G, H, L, M, N, Q, R, S, T, V, or Y, Z8 is A, G, I, K, P, Q, R, S, or T, and, Z9 is F, H, or Y.

[0027] In some embodiments, the third domain (C) comprises a humanized antibody or an antigen-binding fragment thereof. In some embodiments, the third domain (C) comprises a single-domain antibody, VHH domain, scFv, VH domain, VL domain, Fab, Fab’, non-Ig domain, ligand, notchin, or small molecule entity that specifically binds to DLL3. In some embodiments, the third domain (C) comprises a single-domain antibody. In some embodiments, the second domain (B) binds to bulk serum proteins. In some embodiments, the second domain (B) comprises a single-domain antibody, VHH domain, scFv, VH domain, VL domain, Fab, Fab’, non-Ig domain, ligand, notchin, or small molecule entity that specifically binds to bulk serum proteins. In some embodiments, the second domain (B) comprises a single-domain antibody that specifically binds to bulk serum proteins. In some embodiments, the bulk serum proteins comprise albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, pentameric IgM, or Igκ free light chain. In some embodiments, the bulk serum proteins comprise albumin. In some embodiments, the second domain (B) comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID No. 1769-1778. In some embodiments, the second domain (B) comprises a sequence that is at least about 75% identical to SEQ ID No. 1774.

[0028] In some embodiments, the first domain (A) comprises a single-domain antibody, VHH domain, scFv, VH domain, VL domain, Fab, Fab’, non-Ig domain, ligand, notchin, or small molecule entity that specifically binds to CD3. In some embodiments, the first domain (A) comprises a sequence selected from the group consisting of SEQ ID No. 1793-1807. In some embodiments, the third domain (C) comprises the following formula: f1-r1-f2-r2-f3-r3-f4 Here, r1 is identical to or includes one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 443-884 and 1887, r2 is identical to or includes one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 885-1326 and 1888, and r3 is identical to or includes one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889, where f1, f2, f3, and f4 are framework residues. In some embodiments, the third domain (C) includes a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886. In some embodiments, domain C and B are connected by linker L1, and domain B and A are connected by linker L2. In some embodiments, linkers L1 and L2 are each independently (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), or (GGGGS) n(SEQ ID No. 1814), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, linkers L1 and L2 are each independently (GGGGS)4 (SEQ ID No. 1817) or (GGGGS)3 (SEQ ID No. 1818). In some embodiments, the protein binds to DLL3 with a binding affinity (Kd) of about 0.1 nM to about 50 nM. In some embodiments, the protein binds to human DLL3, cynomolgus monkey DLL3, or both human and cynomolgus monkey DLL3. In some embodiments, the protein is less than about 80 kDa. In some embodiments, the protein is about 50 to about 75 kDa. In some embodiments, the protein is less than about 60 kDa. In some embodiments, the protein has a half-life of at least about 50 hours. In some embodiments, the protein has a half-life of at least about 100 hours. In some embodiments, the protein has increased tissue penetration compared to IgG to the same DLL3. In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH.

[0029] One embodiment provides a pharmaceutical composition comprising (i) a DLL3-targeted trispecific protein according to any one of the above embodiments, or a DLL3-binding protein according to the present disclosure, and (ii) a pharmaceutically acceptable carrier. One embodiment provides a method for preparing a DLL3-targeted trispecific protein according to any one of the above embodiments, the method comprising: i) providing a DLL3 protein or a fragment thereof; ii) exposing the DLL3 protein or a fragment thereof to a recombinant library of DLL3-binding proteins; iii) selecting from the library a DLL3-binding protein that specifically binds to the oligomer or a derivative thereof; and (iv) using the DLL3-binding protein identified in step (iii) to prepare a DLL3-targeted trispecific protein. In some embodiments, the recombinant library of DLL3-binding proteins is exposed to the DLL3 protein in vitro by screening the recombinant library with the DLL3 protein. In some embodiments, the recombinant library is expressed on the surface of a bacteriophage. In some embodiments, the recombinant library is expressed on the surface of yeast cells. In some embodiments, the recombinant library is expressed on the surface of bacterial cells. In some embodiments, the recombinant library is expressed as an RNA-protein fusion. In some embodiments, the recombinant library is a scFv library or a Fab library. In some embodiments, the recombinant antibody library is a single-domain library.

[0030] One embodiment provides a process for the production of a DLL3-targeted trispecific protein according to any one of the above embodiments, the process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding a DLL3-targeted trispecific protein according to any one of the above embodiments under conditions that allow expression of the DLL3-targeted trispecific protein, and recovering and purifying the protein produced from the culture.

[0031] One embodiment provides a DLL3-binding protein comprising the following formula: f1-r1-f2-r2-f3-r3-f4 wherein r1 is Complementary Determining Region 1 (CDR1) and is identical to or comprises one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID No. 443-884 and 1887; r2 is CDR2 and is identical to or comprises one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID No. 885-1326 and 1888; and r3 is CDR3 and is identical to or comprises one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID No. 1327-1768 and 1889, wherein f1, f2, f3, and f4 are framework residues. In some embodiments, the DLL3-binding protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID No. 1-442 and 1886. In some embodiments, the DLL3-binding protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID No. 1-52. In some embodiments, the DLL3-binding protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID No. 53-86. In some embodiments, the DLL3-binding protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID No. 87-367. In some embodiments, the DLL3-binding protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID No. 368-442. One embodiment provides a DLL3-binding protein derived from a parental DLL3-binding protein comprising the sequence of SEQ ID No. 68. One embodiment provides a DLL3-binding protein derived from a parental DLL3-binding protein comprising the sequence of SEQ ID No. 75 or comprising the sequence of SEQ ID No. 75.

[0032] One embodiment provides a method for preparing a DLL3-binding protein according to the present disclosure, the method comprising: i) providing a DLL3 protein or a fragment thereof; ii) exposing the DLL3 protein or the fragment thereof to a recombinant library of DLL3-binding proteins; and iii) selecting from the library a DLL3-binding protein that specifically binds to the oligomer or a derivative thereof. In some embodiments, the recombinant library of DLL3-binding proteins is exposed to the DLL3 protein in vitro by screening the recombinant library using the DLL3 protein. In some embodiments, the recombinant library is expressed on the surface of a bacteriophage. In some embodiments, the recombinant library is expressed on the surface of a yeast cell. In some embodiments, the recombinant library is expressed on the surface of a bacterial cell. In some embodiments, the recombinant library is expressed as an RNA-protein fusion. In some embodiments, the recombinant library is a scFv library or a Fab library. In some embodiments, the recombinant antibody library is a single-domain library.

[0033] One embodiment provides a process for the production of a DLL3-binding protein according to the present disclosure, the process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding a DLL3-binding protein according to the present disclosure under conditions that allow expression of the DLL3-binding protein, and recovering and purifying the protein produced from the culture.

[0034] One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, the method comprising administering to a subject a DLL3-targeted trispecific protein according to any one of the above embodiments, a DLL3-binding protein according to the present disclosure, or a pharmaceutical composition as provided herein. In some embodiments, the subject is human. In some embodiments, the method further comprises administering an agent in combination with a DLL3-targeted trispecific protein according to any one of the above embodiments, a DLL3-binding protein according to the present disclosure, or a pharmaceutical composition as provided herein. In some embodiments, the DLL3-targeted trispecific protein or DLL3-binding protein selectively binds to tumor cells expressing DLL3. In some embodiments, the DLL3-targeted trispecific protein mediates T cell killing of tumor cells expressing DLL3. In some embodiments, the neoplastic disease is a solid tumor disease. In some embodiments, the solid tumor disease includes lung cancer, gastric cancer, ovarian cancer, or triple negative breast cancer. In some embodiments, the solid tumor disease is metastatic.

[0035] One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, the method comprising administering a DLL3-targeted trispecific protein comprising a DLL3-binding domain comprising a sequence selected from the group consisting of SEQ ID No. 1-442 and 1886, or a DLL3-binding protein comprising a sequence selected from the group consisting of SEQ ID No. 1-442 and 1886. In some embodiments, the DLL3-targeted trispecific protein or DLL3-binding protein selectively binds to tumor cells expressing DLL3. In some embodiments, the DLL3-targeted trispecific protein induces T cell killing of tumor cells expressing DLL3. In some embodiments, the neoplastic disease is a solid tumor disease. In some embodiments, the solid tumor disease includes lung cancer, gastric cancer, ovarian cancer, or triple negative breast cancer. In some embodiments, the solid tumor disease is metastatic.

[0036] One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, the method comprising administering a DLL3 binding domain comprising the sequence set forth in SEQ ID No. 68 or 75, or a DLL3 binding protein comprising the sequence set forth in SEQ ID No. 68 or 75. In some embodiments, the DLL3-targeted trispecific protein or DLL3 binding protein is at a dosage of up to 10 mg / kg. In some embodiments, the protein is administered at least once a week. In some embodiments, the protein is administered twice a week. In some embodiments, the protein is administered once every two weeks. In some embodiments, the protein is administered once every three weeks.

[0037] In one embodiment, a DLL3 binding protein is provided that comprises the amino acid sequence set forth in SEQ ID No. 1890 or SEQ ID No. 1891.

[0038] One embodiment provides a DLL3-targeted trispecific protein, the DLL3-targeted trispecific protein comprising (a) a first domain (A) that is a single-chain variable fragment that specifically binds to human CD3, (b) a second domain (B) that is a single-domain antibody that specifically binds to human serum albumin protein, (c) a third domain (C) that is a single-domain antibody that specifically binds to DLL3 protein, wherein the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 1-442 and 1886.

[0039] In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, H2N-(C)-(B)-(A)-COOH, or H2N-(C)-(A)-(B)-COOH, or, by linkers L1 and L2, in the order of H2N-(A)-L1-(B)-L2-(C)-COOH, H2N-(A)-L1-(C)-L2-(B)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2N-(B)-L1-(C)-L2-(A)-COOH, H2N-(C)-L1-(B)-L2-(A)-COOH, or H2N-(C)-L1-(A)-L2-(B)-COOH.

[0040] In some embodiments, the third domain (C) is an affinity matured binding molecule derived from a parent molecule that specifically binds to the DLL3 protein. In some embodiments, the affinity matured binding DLL3 molecule has a binding affinity for the DLL3 protein that is about 2-fold, about 50-fold greater than the binding affinity of the parent molecule for the DLL3 protein.

[0041] In some embodiments, the third domain (C) comprises a sequence selected from the group consisting of SEQ ID No. 1-442 and 1886. In some embodiments, the third domain (C) comprises a sequence selected from the group consisting of SEQ ID No. 1-52. In some embodiments, the third domain (C) comprises CDR1, CDR2, and CDR3, where CDR1 comprises a sequence selected from the group consisting of SEQ ID No. 443-884 and 1887, where CDR2 comprises a sequence selected from the group consisting of SEQ ID No. 885-1326 and 1888, and where CDR3 comprises a sequence selected from the group consisting of SEQ ID No. 1327-1768 and 1889. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID No. 495-528. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID No. 937-970. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID No. 1379-1412. In some embodiments, the third domain (C) comprises a sequence selected from the group consisting of SEQ ID No. 53-86.

[0042] In some embodiments, the third domain (C) includes CDR1, CDR2, and CDR3, and here, CDR1 includes a sequence selected from the group consisting of SEQ ID No. 529-809. In some embodiments, CDR2 includes a sequence selected from the group consisting of SEQ ID No. 971-1251. In some embodiments, CDR3 includes a sequence selected from the group consisting of SEQ ID No. 1379-1412. In some embodiments, the third domain (C) includes a sequence selected from the group consisting of SEQ ID No. 87-367. In some embodiments, the third domain (C) includes CDR1, CDR2, and CDR3, and here, CDR1 includes a sequence selected from the group consisting of SEQ ID No. 810-884. In some embodiments, CDR2 includes a sequence selected from the group consisting of SEQ ID No. 1252-1326. In some embodiments, CDR3 includes a sequence selected from the group consisting of SEQ ID No. 1692-1768. In some embodiments, the third domain (C) includes a sequence selected from the group consisting of SEQ ID No. 368-442. In some embodiments, the first domain (A) includes a sequence selected from the group consisting of SEQ ID No. 1793-1807 and 1897-1898.

[0043] In some embodiments, the second domain (B) includes a sequence selected from the group consisting of SEQ ID No. 1769-1778. In some embodiments, linkers L1 and L2 are each independently (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), or (GGGGS) n(SEQ ID No. 1814), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH or, by linkers L1 and L2, in the order of H2N-(A)-L1-(B)-L2-(C)-COOH. In some embodiments, linkers L1 and L2 independently comprise the sequence GGGGSGGGS (SEQ ID No. 1808). In some embodiments, the DLL3-targeted trispecific protein comprises the sequence of SEQ ID No. 1890 or SEQ ID No. 1891.

[0044] One embodiment provides a DLL3-targeted trispecific protein, wherein the DLL3-targeted trispecific protein comprises: (a) a first domain (A) that is a single-chain variable fragment specifically binding to human CD3; (b) a second domain (B) that is a single-domain antibody specifically binding to human serum albumin protein; and (c) a third domain (C) that is a single-domain antibody specifically binding to DLL3, where the third domain comprises the sequence of SEQ ID No. 68 or SEQ ID No. 75, or is derived from SEQ ID No. 68 or SEQ ID No. 75. In some embodiments, the third domain (C) is derived from SEQ ID No. 68 or SEQ ID No. 75. In some embodiments, the third domain (C) comprises the sequence of SEQ ID No. 68 or the sequence of SEQ ID No. 75.

[0045] One embodiment provides a DLL3-binding protein comprising the following formula: f1-r1-f2-r2-f3-r3-f4 Here, r1 is Complementary Determining Region 1 (CDR1) and is identical to or includes one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID No. 443-884 and 1887, r2 is CDR2 and is identical to or includes one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID No. 885-1326 and 1888, and r3 is CDR3 and is identical to or includes one or more amino acid residue substitutions relative to a sequence selected from the group consisting of SEQ ID No. 1327-1768 and 1889, where f1, f2, f3, and f4 are framework residues. In some embodiments, CDR1 includes a sequence selected from the group consisting of SEQ ID No. 443-884 and 1887. Here, CDR2 includes a sequence selected from the group consisting of SEQ ID No. 885-1326 and 1888, and here, CDR3 includes a sequence selected from the group consisting of SEQ ID No. 1327-1768 and 1889.

[0046] One embodiment provides a DLL3-targeted trispecific protein, wherein the DLL3-targeted trispecific protein (a) a first domain (A) that is a single-chain variable fragment specifically binding to human CD3, (b) a second domain (B) that is a single-domain antibody specifically binding to human serum albumin protein, (c) a third domain (C) that is a single-domain antibody specifically binding to DLL3 protein, wherein the third domain (C) includes a CDR1 having the amino acid sequence of SEQ ID No. 874, a CDR2 having the amino acid sequence of SEQ ID No. 1316, and a CDR3 having the amino acid sequence of SEQ ID No. 1758.

[0047] One embodiment provides a DLL3-targeted trispecific protein, wherein the DLL3-targeted trispecific protein (a) A first domain (A) which is a single-chain variable fragment that specifically binds to human CD3, (b) A second domain (B) which is a single-domain antibody that specifically binds to human serum albumin protein, (c) A third domain (C) which is a single-domain antibody that specifically binds to DLL3, wherein the third domain comprises a CDR1 having an amino acid sequence selected from the group consisting of SEQ ID No. 851, 867, 871, 872, 873, 874, and 1887, a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID No. 1293, 1309, 1313, 1314, 1315, 1316, and 1888, and a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID No. 1735, 1751, 1755, 1756, 1757, 1758, and 1889. In some embodiments, the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 408, 425, 432, 430, 431, and 1886. In some embodiments, the third domain (C) comprises a sequence selected from the group consisting of SEQ ID No. 408, 425, 432, 430, 431, and 1886.

[0048] In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, H2N-(C)-(B)-(A)-COOH, or H2N-(C)-(A)-(B)-COOH, or, by linkers L1 and L2, in the order of H2N-(A)-L1-(B)-L2-(C)-COOH, H2N-(A)-L1-(C)-L2-(B)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2N-(B)-L1-(C)-L2-(A)-COOH, H2N-(C)-L1-(B)-L2-(A)-COOH, or H2N-(C)-L1-(A)-L2-(B)-COOH.

[0049] In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH or, by linkers L1 and L2, in the order of H2N-(A)-L1-(B)-L2-(C)-COOH. In some embodiments, the domains are linked in the order of H2N-(C)-(B)-(A)-COOH or, by linkers L1 and L2, in the order of H2N-(C)-L1-(B)-L2-(A)-COOH. In some embodiments, the third domain (C) is an affinity matured binding molecule derived from a parent molecule that specifically binds to the DLL3 protein.

[0050] In some embodiments, the affinity matured binding DLL3 molecule has a binding affinity for the DLL3 protein that is about 2-fold, about 50-fold greater than the binding affinity of the parent molecule for the DLL3 protein. In some embodiments, the parent molecule comprises the amino acid sequence of SEQ ID No. 68 or SEQ ID No. 75.

[0051] In some embodiments, linkers L1 and L2 are each independently (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), or (GGGGS) n (SEQ ID No. 1814) selected from the group consisting of, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, linkers L1 and L2 independently comprise the sequence of GGGGSGGGS (SEQ ID No. 1808).

[0052] In some embodiments, the second domain (B) comprises an amino acid sequence selected from the group consisting of SEQ ID No. 1769-1778. In some embodiments, the first domain (A) comprises an amino acid sequence selected from the group consisting of SEQ ID No. 1793-1802 and 1897-1898. In some embodiments, the DLL3-targeted trispecific protein comprises the sequence of SEQ ID No. 1890 or SEQ ID No. 1891. In some embodiments, the third domain (C) binds to a human DLL3 protein comprising the sequence of SEQ ID NO. 1893.

[0053] One embodiment provides a DDL3-binding protein comprising a CDR1 having the amino acid sequence of SEQ ID No. 874, a CDR2 having the amino acid sequence of SEQ ID No. 1316, and a CDR3 having the amino acid sequence of SEQ ID No. 1758. In some embodiments, the DLL3-binding protein comprises a sequence that is at least 80% identical to the amino acid sequence of SEQ ID No. 432.

[0054] One embodiment provides a DDL3-binding protein comprising a CDR1 having an amino acid sequence selected from the group consisting of SEQ ID No. 851, 867, 871, 872, 873, 874, and 1887, a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID No. 1293, 1309, 1313, 1314, 1315, 1316, and 1888, and a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID No. 1735, 1751, 1755, 1756, 1757, 1758, and 1889. In some embodiments, the DLL3-binding protein comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID No. 408, 425, 432, 430, 431, and 1886.

[0055] One embodiment provides a method of treating or ameliorating a disease, comprising administering an effective amount of a DLL3-targeted trispecific protein according to any one of the above embodiments.

[0056] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby 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.

Brief Description of the Drawings

[0057] The novel features of the invention are set forth with particularity in the appended claims. To better understand the features and advantages of the invention, reference should be made to the following detailed description, which illustrates exemplary embodiments in which the principles of the invention are used, and the accompanying drawings.

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BRIEF DESCRIPTION OF THE INVENTION

[0058] In some embodiments, proteins that specifically bind to delta-like ligand 3 (DLL3), multispecific (e.g., trispecific) proteins containing the above proteins, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making the above proteins are described herein. Further provided are methods of using at least one of the disclosed DLL3-binding proteins, or DLL3-targeted trispecific proteins comprising the above, in the prevention and treatment of diseases, disorders, and ailments. The DLL3-targeted trispecific protein can specifically bind to DLL3, similar to CD3, and has a half-life extension domain, such as a domain that can specifically bind 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 includes an antibody, such as a trispecific antibody.

[0059] Specific Definitions An "antibody" typically refers to a Y-shaped tetrameric protein containing two heavy (H) and two light (L) polypeptide chains held together by shared disulfide bonds and non-covalent interactions. Human light chains contain a variable domain (VL) and a constant domain (CL), and the constant domain can be readily classified as kappa or lambda based on amino acid sequence and gene locus. Each heavy chain contains one variable domain (VH) and a constant region, which, in the case of IgG, IgA, and IgD, contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the classification of IgG, IgA, and IgD, the domains of CH1 and CH2 are separated by a flexible hinge region, which is a variable-length proline- and cysteine-rich segment (usually about 10 to about 60 amino acids in IgG). The variable domains of both the light and heavy chains are joined to the constant domain by a "J" region of about 12 or more amino acids, and the heavy chain further has a "D" region of about 10 additional amino acids. Each class of antibody contains interchain and intrachain disulfide bonds formed by paired cysteine residues. Immunoglobulin molecules have two types of native disulfide bridges or bonds: interchain and intrachain disulfide bonds. The position and number of interchain disulfide bonds vary depending on the class and species of immunoglobulin. Interchain disulfide bonds are located on the surface of the immunoglobulin, are accessible to the solvent, and are usually relatively easily reduced. In the human IgG1 isotype, there are four interchain disulfide bonds, one from each heavy chain to a light chain and two between the heavy chains. Interchain disulfide bonds are not necessary for chain association. As is well known, the cysteine-rich IgG1 hinge region of the heavy chain is generally maintained as consisting of three parts: an upper hinge, a core hinge, and a lower hinge. Those skilled in the art will understand that the IgG1 hinge region contains cysteines within the heavy chain that include interchain disulfide bonds (two heavy / heavy, two heavy / light), which provide the structural flexibility that promotes the movement of the Fab. The interchain disulfide bond between the light and heavy chains of IgG1 is formed between C214 of the kappa or lambda light chain and C220 within the upper hinge region of the heavy chain.The inter-chain disulfide bonds between the heavy chains are located at positions C226 and C229 (all numbered according to EU indices following Kabat, et al. below).

[0060] As used herein, the term “antibody” includes polyclonal, multi-clonal, monoclonal, chimeric, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, immunospecific antibody fragments, e.g., Fd, Fab, F(ab’)2, F(ab’) fragments, single-chain fragments (e.g., ScFv and ScFvFc), disulfide-linked Fvs (sdFv), Fd fragments consisting of the VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAb (VH, VL, or VHH domains), as well as derivatives thereof including Fc fusions and other modifications, and any other immunoreactive molecule, so long as it contains a binding site for preferential association or binding with the DLL3 protein. Further, unless otherwise specified by context, the term further includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The heavy-chain constant domains corresponding to the various classes of antibodies are typically designated by the corresponding lower-case Greek letters α, δ, ε, γ, and μ, respectively. The light chains of antibodies of any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain.

[0061] In some embodiments, the DLL3 binding domain of the DLL3-targeted trispecific proteins of the present disclosure includes an antibody of only the heavy chain, such as a VH or VHH domain. In some cases, the DLL3 binding protein includes an antibody of only the heavy chain that is 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-targeted trispecific proteins of the present disclosure includes a VHH. The term "VHH" as used herein refers to a single-chain antibody binding domain lacking a light chain. In some cases, the VHH is derived from an antibody of the type found in camels or cartilaginous fish that are naturally lacking a light chain, or can be derived from a synthetic non-immune VHH constructed accordingly. Each heavy chain includes a variable region encoded by V, D, and J exons. The VHH is, in some cases, a native VHH, such as a VHH from camels, or a recombinant protein comprising a heavy chain variable domain. In some embodiments, the VHH is derived from a species selected from the group consisting of camels, llamas, vicuñas, guanacos, and cartilaginous fish (including but not limited to sharks). In another embodiment, the VHH is derived from alpacas (including but not limited to Huacaya Alpaca and Suri alpaca).

[0062] As used herein, the term "variable region" or "variable domain" refers to the fact that certain portions of the variable domains vary widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed throughout the variable domains of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the variable domains of the light chain (VL) and the heavy chain (VH). The more highly conserved portions of the variable domains are called frameworks (FRs). The native variable domains of the heavy and light chains each contain four FR regions that mostly adopt a β-sheet conformation, are joined by the three CDRs, form loop junctions and, in some cases, form part of the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains do not directly participate in binding the antibody to an antigen, but exhibit various effector functions such as the involvement of the antibody in antibody-dependent cytotoxicity. In some cases, ScFv fragments (for variable single-chain fragments) obtained by genetic engineering associate in a single polypeptide chain with the VH and VL regions of the antibody separated by a peptide linker.

[0063] In some embodiments of the present disclosure, it includes a DLL3 binding domain such as the DLL3 binding domain of a DLL3-targeted trispecific protein, a single-domain antibody such as an antibody of only the heavy chain such as the domain of VH or VHH, and includes three CDRs. Such an antibody of only the heavy chain binds to DLL3 as a monomer that does not depend on dimerization with the VL (variable light chain) region for optimal binding affinity in some embodiments. In some embodiments of the present disclosure, the CD3 binding domain of the DLL3-targeted trispecific protein includes an scFv. In some embodiments of the present disclosure, the albumin binding domain of the DLL3-targeted trispecific protein includes an antibody of only the heavy chain such as a single-domain antibody including a VH domain or a VHH domain.

[0064] The amino acid assignments for each domain, framework region, and CDR follow, in some embodiments, one of the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Ed.), US Dept. of Health and Human Services, PHS, NIH, NIH Publication No.91-3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3rd Ed., Wily-VCH Verlag GmbH and Co or AbM (Oxford Molecular / MSI Pharmacopia), unless otherwise specified. It is not intended that the CDRs of the present disclosure necessarily correspond to the Kabat numbering convention. In some embodiments of the present disclosure, the DLL3-binding protein includes a single domain antibody such as an antibody of only the heavy chain, such as a VH or VHH domain, and includes three CDRs. Such an antibody of only the heavy chain binds DLL3 as a monomer that, in some embodiments, does not rely on dimerization with a VL (variable light chain) region for optimal binding affinity.

[0065] "Numbering of variable domain residues as in Kabat" or "numbering of amino acid positions as in Kabat", and variations thereof, refer to the numbering system used for the heavy chain variable domain or the light chain variable domain of the antibody compilation in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the omission or insertion of FR or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2, and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment in the homologous region of the antibody sequence with the sequence having the "standard" Kabat numbering.

[0066] The term "framework" or "FR" residue (or region) refers to variable domain residues other than those of the CDR or hypervariable region as defined herein. "Human consensus framework" is a framework that represents the amino acid residues that most commonly occur upon selection of the human immunoglobulin VL or VH framework sequence.

[0067] As used herein, the term "percent (%) amino acid sequence identity" with respect to an array is defined as the percentage of amino acid residues in a candidate array that are identical to the amino acid residues in a particular array after aligning the arrays to achieve maximum percent sequence identity, introducing gaps as necessary, and without considering any conservative substitutions as part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved by various methods within the art, such as publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0068] As used herein, "elimination half-life" is used in its ordinary meaning as described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, this term is meant to encompass a quantitative measure of the passage of time of drug elimination. Since drug concentrations usually do not reach the concentrations necessary to saturate the elimination process, the elimination of most drugs is exponential (i.e., follows first-order kinetics). The rate of an exponential process can be expressed by its rate constant k, which represents the fractional rate of change per unit of time, or by its half-life t1 / 2, which is the time required for 50% of the process to be completed. The units of these two constants are time−1 and time, respectively. The first-order reaction rate constant and the half-life of a reaction are simply related (k × t1 / 2 = 0.693) and can be interchanged as appropriate. Since first-order elimination kinetics dictate that a constant fraction of the drug is lost per unit of time, a plot of the logarithm of drug concentration versus time is linear at all times after the initial distribution phase (i.e., after drug absorption and distribution are complete). The half-life of drug elimination can be accurately determined from such a graph.

[0069] As used herein, the term "binding affinity" refers to the affinity of the proteins described in the present disclosure for a binding target, and is expressed numerically using the "Kd" value. If two or more proteins are shown to have equivalent binding affinity for their binding target, the Kd values for the binding of each protein to the binding target are within ±2-fold of each other. If two or more proteins are shown to have equivalent binding affinity for a single binding target, the Kd values for the binding of each protein to the single binding target are within ±2-fold of each other. If a protein is shown to bind to two or more targets with equivalent binding affinity, the Kd values for the binding of the protein to the two or more targets are within ±2-fold of each other. Generally, a high Kd value corresponds to weak binding. In some embodiments, "Kd" is measured by a radiolabeled antigen binding assay (RIA) or surface plasmon resonance assay using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.). In one embodiment, "on-rate" or "rate of association" or "kon", and "off-rate" or "rate of dissociation" or "koff" are also determined by surface plasmon resonance technology using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.). In further embodiments, "Kd", "kon", and "koff" are measured using an OCTET® system (Pall Life Sciences).In an exemplary method of measuring binding affinity using OCTET® Systems, a ligand, e.g., biotinylated human or cynomolgus DLL3, is immobilized on the surface of an OCTET® streptavidin capillary sensor tip, and then the tip of the streptavidin is activated according to the manufacturer's instructions using human or cynomolgus DLL3 protein at about 20 - 50 μg / ml. A solution of PBS / casein is also introduced as a blocking agent. For measuring the association reaction rate, DLL3-binding protein variants are introduced at concentrations in the range of about 10 ng / mL to about 100 μg / mL, about 50 ng / mL to about 5 μg / mL, or about 2 ng / mL to about 20 μg / mL. In some embodiments, the DLL3-binding single domain protein is used at a concentration in the range of about 2 ng / mL to about 20 μg / mL. Complete dissociation is observed in the case of an assay buffer without the binding protein, which is the negative control. Thereafter, the kinetic parameters of the binding reaction are determined using an appropriate tool, e.g., ForteBio software.

[0070] One embodiment provides a DLL3-binding protein (also referred to herein as a DLL3-binding domain, such as the DLL3-binding domain of the DLL3 trispecific antibody of the present disclosure), which comprises a CDR1 sequence comprising a sequence selected from the group consisting of SEQ ID No. 443 - 884 and 1887, a CDR2 sequence comprising a sequence selected from the group consisting of SEQ ID No. 885 - 1326 and 1888, and a CDR3 sequence comprising a sequence selected from the group consisting of SEQ ID No. 1327 - 1768 and 1889, and includes a single domain antibody. In some embodiments, the DLL3-binding protein of the present disclosure is quite small, and in some embodiments, it is contemplated to be 25 kDa or less, 20 kDa or less, 15 kDa or less, or 10 kDa or less. In certain examples, EGFR binding is 5 kDa or less when it is a peptide or small molecule entity.

[0071] In one aspect, a DLL3-targeting trispecific protein (also referred to herein as a DLL3-binding trispecific protein, a DLL3 trispecific protein, or a DLL3 TriTAC™) comprises (a) a first domain (A) that specifically binds to human CD3, (b) a second domain (B) that is a half-life extension domain, and (c) a third domain (C) that specifically binds to DLL3. The three domains in the DLL3-targeting trispecific protein are arranged in any order. Thus, it is contemplated that the domain order of the DLL3-targeting trispecific protein is as follows: H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, H2N-(C)-(B)-(A)-COOH, or H2N-(C)-(A)-(B)-COOH.

[0072] In some embodiments, the DLL3-targeting trispecific protein has a domain order of H2N-(A)-(B)-(C)-COOH. In some embodiments, the DLL3-targeting trispecific protein has a domain order of H2N-(A)-(C)-(B)-COOH. In some embodiments, the DLL3-targeting trispecific protein has a domain order of H2N-(B)-(A)-(C)-COOH. In some embodiments, the DLL3-targeting trispecific protein has a domain order of H2N-(B)-(C)-(A)-COOH. In some embodiments, the DLL3-targeting trispecific protein has a domain order of H2N-(C)-(B)-(A)-COOH. In some embodiments, the DLL3-targeting trispecific protein has a domain order of H2N-(C)-(A)-(B)-COOH.

[0073] In some embodiments, the DLL3-targeting trispecific protein has an HSA (also referred to herein as ALB) binding domain as the central domain such that the domain order is H2N-(A)-(B)-(C)-COOH, or H2N-(C)-(B)-(A)-COOH. In such embodiments where the HSA binding domain is the central domain, the binding domains for CD3 and DLL3 are intended to be provided with additional flexibility to bind to their respective targets.

[0074] In some embodiments, the trispecific binding protein comprises a third domain that specifically binds to DLL3, where the third domain is optionally a DLL3-binding single domain antibody, which binds to DLL3 with an affinity equal to or greater than that of a reference DLL3-binding parental molecule. The third domain, in some embodiments, comprises an affinity matured DLL3-binding molecule (e.g., an affinity matured DLL3-binding single domain antibody), and is derived from a DLL3-binding parental molecule that contains one or more amino acid mutations (e.g., stabilizing mutations, destabilizing mutations) relative to the DLL3-binding parental molecule. In some embodiments, the affinity matured DLL3-binding molecule has superior stability to a selected destabilizing agent as compared to the reference DLL3-binding parental molecule. In some embodiments, the affinity matured DLL3-binding molecule is identified by a process that includes panning of one or more pre-candidate DLL3-binding molecules expressed in a phage display library against a DLL3 protein such as a human DLL3 protein. The pre-candidate DLL3-binding molecules, in some embodiments, contain amino acid substitutions in the variable region, CDRs, or framework residues relative to the parental molecule.

[0075] As used herein, "phage display" is a technique in which a mutant polypeptide is displayed as a fusion protein with respect to at least a part of the coat protein on the surface of a phage, filamentous phage, or particle. The usefulness of phage display lies in the fact that a large library of randomized protein mutants can be rapidly and efficiently selected for sequences that bind to a target molecule with high affinity. Displaying libraries of peptides and proteins on phages has been used to screen for those with specific binding properties from among millions of polypeptides. The multivalent phage display method is used to display small random peptides and small proteins by fusing them to either gene III or gene VIII of the filamentous phage. Wells and Lowman, Curr. Opin. Struct. Biol, 3:355-362 (1992) and the references cited therein. In monovalent phage display, a protein or peptide library is fused to gene III or a part thereof and expressed at a low level in the presence of the wild-type gene III protein, so that the phage particles display one copy of the fusion protein or no fusion protein at all. The avidity effect is smaller compared to multivalent phages, and the selection is based on the affinity for the endogenous ligand. Phagemid vectors are used, which facilitates DNA manipulation. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991)

[0076] In some embodiments, panning involves using varying binding times and concentrations to identify DLL3-binding molecules with increased or decreased on-rates from pre-candidate DLL3-binding molecules. In some embodiments, panning involves using varying wash times to identify DLL3-binding molecules with increased or decreased on-rates from pre-candidate DLL3-binding molecules. In some embodiments, panning involves using varying binding times and varying wash times. In some embodiments, one or more stabilizing mutations are combined to enhance the stability of affinity matured DLL3-binding molecules, for example, by shuffling to create a second stage combinatorial library from such mutants, a second round of panning, and then performing binding selection.

[0077] In some embodiments, the affinity matured DLL3-binding molecule has an affinity for a DLL3 protein (such as a human DLL3 protein) that is equal to or greater than that of the DLL3-binding parental molecule, which has reduced cross-reactivity with selected substances such as ligands, proteins, antigens, etc., other than the DLL3 epitope to which the DLL3-binding parental molecule is specific or is designed to be specific, or in some embodiments, increased cross-reactivity. With respect to the latter, in some embodiments, testing in animal models is more successful when the affinity matured DLL3-binding molecule is reacted with both human DLL3 and mouse DLL3 or cynomolgus monkey DLL3, which are the corresponding targets in the animal models. In some embodiments, the parental DLL3-binding molecule binds to human DLL3 with an affinity of about 10 nM or less and to cynomolgus monkey DLL3 with an affinity of about 15 nM or less. In some embodiments, the affinity matured DLL3-binding molecule identified after one round of panning binds to human DLL3 with an affinity of about 5 nM or less and to cynomolgus monkey DLL3 with an affinity of about 7.5 nM or less. In some embodiments, the affinity matured DLL3-binding molecule identified after two rounds of panning binds to human DLL3 with an affinity of about 2.5 nM or less and to cynomolgus monkey DLL3 with an affinity of about 3.5 nM or less.

[0078] In some embodiments, domains A, B, and C of the trispecific binding proteins of the present disclosure are each independently an antigen-specific binding domain polypeptide that specifically binds to a target such as a target on a diseased cell, or a target on another cell that supports a disease state, e.g., 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, the antigen-specific binding domain includes an antibody, a heavy-chain only antibody including a single-chain antibody, Fabs, Fvs, T cell receptor binding domains, ligand binding domains, receptor binding domains, domain antibodies, single-domain antibodies, minibodies, nanobodies, peptibodies, or various other antibody mimics (such as affimers, affitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, knotted domain-based proteins, avimers, nottins, finomers, darpins, afibodies, affilins, monobodies, and armadillo repeat protein-based proteins, etc.).

[0079] In some embodiments, the DLL3-targeting trispecific protein described herein comprises a DLL-binding polypeptide having a sequence selected from SEQ ID No. 1-442 and 1886, a subsequence thereof, and a variant thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3-binding polypeptide (i.e., the third domain (C)) having at least 70%-95% or more homology to a sequence selected from SEQ ID No. 1-442 and 1886, a subsequence thereof, and a variant thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3-binding polypeptide (i.e., the 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. 1-442 and 1886, a subsequence thereof, and a variant thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3-binding polypeptide (i.e., the third domain (C)) having at least 70%-95% or more identity to a sequence selected from SEQ ID No. 1-442 and 1886, a subsequence thereof, and a variant thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3-binding polypeptide (i.e., the third domain (C)) having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from the group consisting of SEQ ID No. 1-442 and 1886, a subsequence thereof, and a variant thereof.

[0080] The DLL3-targeted trispecific proteins described herein are designed to be able to specifically target cells expressing DLL3 by recruiting cytotoxic T cells. In some embodiments, this improves efficacy compared to ADCC (antibody-dependent cell cytotoxicity), which uses full-length antibodies targeting a single antigen and cannot directly recruit cytotoxic T cells. In contrast, by binding to CD3 molecules specifically expressed on these cells, the DLL3-targeted trispecific proteins can crosslink cytotoxic T cells to cells expressing DLL3 in a very specific manner, thereby directing the potential cytotoxicity of the T cells towards the target cells. The DLL3-targeted trispecific proteins described herein bind to cytotoxic T cells by binding to surface-expressed CD3 proteins that form part of the TCR. Simultaneous binding of multiple DLL3 trispecific antigen-binding proteins to CD3 and DLL3 expressed on the surface of a particular cell causes T cell activation and mediates subsequent lysis of the particular DLL3-expressing cells. Thus, the DLL3-targeted trispecific proteins are intended to exhibit potent, specific, and efficient target cell killing. In some embodiments, the DLL3-targeted trispecific proteins described herein stimulate the killing of target cells by cytotoxic T cells to eliminate pathogenic cells (e.g., tumor cells expressing DLL3). In some of such embodiments, the cells are selectively eliminated, thereby reducing the potential for toxic side effects.

[0081] The DLL3-targeted trispecific proteins described herein further provide advantages over conventional monoclonal antibodies and other smaller bispecific molecules. Generally, the efficacy of recombinant protein pharmaceuticals is highly dependent on the endogenous pharmacokinetics of the protein itself. One advantage here is that the DLL3-targeted trispecific proteins described herein have an extended pharmacokinetic elimination half-life by having a half-life extension domain such as a domain that specifically binds to serum albumin protein (e.g., human serum albumin protein, HSA). In this regard, the DLL3-targeted trispecific proteins described herein have, in some embodiments, an extended serum elimination half-life of about 2, 3, about 5, about 7, about 10, or about 14 days. This is in contrast to other binding proteins such as BiTE or DART molecules that have a relatively very short half-life. For example, the BiTE CD19×CD3 bispecific scFv-scFv fusion molecule requires drug delivery by continuous infusion (intravenous) due to its short half-life. The longer endogenous half-life of the DLL3-targeted trispecific proteins solves this problem, thereby increasing treatment possibilities such as lower dose pharmaceutical formulations, reduced frequency of administration, and / or novel pharmaceutical compositions.

[0082] The DLL3-targeted trispecific proteins described herein further have an optimal size for enhancing tissue penetration and tissue distribution. If the size is large, the penetration or distribution of the protein in the target tissue is limited or hindered. The DLL3-targeted trispecific proteins described herein avoid this by having a small size that enhances tissue penetration and distribution. Accordingly, the DLL3-targeted trispecific proteins described herein, in some embodiments, have a size of about 50 kDa to about 80 kDa, about 50 kDa to about 75 kDa, about 50 kDa to about 70 kDa, or about 50 kDa to about 65 kDa. In some embodiments, the size of the DLL3-targeted trispecific protein is less than about 60 kDa. Thus, the size of the DLL3-targeted trispecific protein is advantageous over an IgG antibody, which is about 150 kDa, and over BiTE and DART bispecific antibody molecules, which are about 55 kDa but have an unextended half-life and are thus rapidly removed by the kidneys.

[0083] In further embodiments, the DLL3-targeted trispecific proteins described herein have a size optimized for enhanced tissue penetration and distribution. In these embodiments, the DLL3-targeted trispecific proteins are constructed to be as small as possible while retaining specificity for their targets. Accordingly, in these embodiments, the DLL3-targeted trispecific proteins described herein have a size of from about 20 kDa to about 40 kDa, or from about 25 kDa to about 35 kDa, ~ about 40 kDa, ~ about 45 kDa, ~ about 50 kDa, ~ about 55 kDa, ~ about 60 kDa, ~ about 65 kDa. In some embodiments, the DLL3-targeted trispecific proteins described herein have a size of about 50 kDa, 49 kDa, 48 kDa, 47 kDa, 46 kDa, 45 kDa, 44 kDa, 43 kDa, 42 kDa, 41 kDa, 40 kDa, about 39 kDa, about 38 kDa, about 37 kDa, about 36 kDa, about 35 kDa, about 34 kDa, about 33 kDa, about 32 kDa, about 31 kDa, about 30 kDa, about 29 kDa, about 28 kDa, about 27 kDa, about 26 kDa, about 25 kDa, about 24 kDa, about 23 kDa, about 22 kDa, about 21 kDa, or about 20 kDa. A typical approach to small size is by using single domain antibody (sdAb) fragments for each of the domains. For example, a particular DLL3 trispecific antigen-binding protein has an anti-CD3 sdAb, an anti-ALB sdAb, and sdAbs for DLL3. This reduces the size of a typical DLL3 trispecific antigen-binding protein to less than 60 kDa. Thus, in some embodiments, all of the domains of the DLL3-targeted trispecific protein are single domain antibody (sdAb) fragments. In some embodiments, the DLL3-binding protein is quite small and, in some embodiments, is contemplated to be 25 kDa or less, 20 kD or less, 15 kDa or less, or 10 kDa or less. In certain instances, the DLL3-binding protein is 5 kDa or less when it is a peptide or small molecule entity.

[0084] In other embodiments, the DLL3-targeted trispecific proteins described herein include small molecule entity (SME) binders for ALB, DLL3, CD3, or all. SME binders are small molecules with an average size of about 500 - 2000 Da and are bound to the DLL3-targeted trispecific proteins by known methods such as sortase ligation or conjugation. In these examples, one of the domains of the DLL3-targeted trispecific antigen-binding protein is the sortase recognition sequence, LPETG (SEQ ID NO:1896). To bind an SME binder to a DLL3-targeted trispecific antigen-binding protein having a sortase recognition sequence, the protein is cultured with sortase and the SME binder, whereby the sortase binds the SME binder to the recognition sequence. In yet other embodiments, the domain of the DLL3-targeted trispecific proteins described herein that binds to DLL3 includes a notch peptide for binding to DLL3. Notch is a disulfide-stabilized peptide with a cysteine knot scaffold and has an average size of about 3.5 kDa. Notch has been contemplated for binding to specific tumor molecules such as DLL3. In a further embodiment, the third domain of the DLL3-targeted trispecific proteins described herein that binds to DLL3 includes the native DLL3 ligand.

[0085] Another feature of the DLL3-targeted trispecific proteins described herein is that they are of a single polypeptide design with flexible binding of domains. This enables the easy production and manufacture of the DLL3-targeted trispecific proteins because such proteins can be encoded by a single cDNA molecule that is easily incorporated into vectors. Further, since the DLL3-targeted trispecific proteins described herein are single monomeric polypeptide chains, there are no problems with chain pairing and no requirements for dimerization. The DLL3-targeted trispecific proteins described herein have a reduced tendency to aggregate, unlike other reported molecules such as bispecific proteins having an Fcγ immunoglobulin domain.

[0086] In the DLL3-targeted trispecific proteins described herein, the domains are, in some embodiments, linked by internal linkers L1 and L2, where L1 links the first and second domains of the DLL3-targeted trispecific protein, and L2 links the second and third domains of the DLL3-targeted trispecific protein. Linkers L1 and L2 have an optimized length and / or amino acid composition. In some embodiments, 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 one example, 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 from about 3 to about 15 consecutive amino acid residues, such as 8, 9, or 10. With respect to the amino acid composition of internal linkers L1 and L2, the peptide is selected by properties that confer flexibility to the DLL3-targeted trispecific protein, do not interfere with the binding domains, and likewise are not resistant to cleavage by proteases. For example, glycine and serine residues generally confer protease resistance. Examples of internal linker L1 suitable for linking to the domains in the DLL3-targeted trispecific protein include, but are not limited to, (GS) n (SEQ ID No.1809), (GGS) n (SEQ ID No.1810), (GGGS) n (SEQ ID No.1811), (GGSG) n (SEQ ID No.1812), (GGSGG) n (SEQ ID No.1813), (GGGGS) n (SEQ ID No.1814), (GGGGG) n (SEQ ID No.1815), or (GGG) n(SEQ ID No. 1816) is exemplified, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the internal linker L1 and / or L2 is (GGGGS)4 (SEQ ID No. 1817) or (GGGGS)3 (SEQ ID No. 1818). In another embodiment, the internal linker L1 and / or L2 is GGGGSGGGS (SEQ ID No. 1808).

[0087] In some cases, the domains within the DLL3-targeting trispecific protein are conjugated using an enzyme site-specific conjugation method that includes the use of mammalian or bacterial transglutaminase enzymes. Microbial transglutaminase (mTG) is a highly versatile tool in modern research and biotechnology. Due to the relatively pure enzyme being available in large quantities, its ease of use, and the lack of regulation by calcium and guanosine-5'-triphosphate (GTP), mTG has become a major crosslinking enzyme used in the food industry and the field of biotechnology. Currently, mTG is used in many applications to bind 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).

[0088] In some examples, DLL3-targeted trispecific proteins are provided, where one of the domains includes an acceptor glutamine in the constant region, which can be coupled to another domain via a lysine-based linker (e.g., any primary amine chain that is a substrate of TGase, including alkylamine, oxoamine), where the coupling occurs only on one or more acceptor glutamine residues present in a targeting moiety outside the antigen-binding site (e.g., outside the variable region, within the constant region). Thus, conjugation does not occur on glutamines within the variable region, i.e., on glutamines where at least part of the surface is exposed. The trispecific protein is, in some examples, formed by reacting one of the domains with a lysine-based linker in the presence of TGase.

[0089] In some embodiments, when one or more domains within the DLL3-targeted triple-binding protein are directly linked, a hybrid vector is made and the DNAs encoding the directly linked domains are ligated directly to each other. In some embodiments, when a linker is used, a hybrid vector is made, where the DNA encoding the first of the three domains is ligated to the DNA encoding one end of the first linker portion, and the DNA encoding the second of the three domains is ligated to the other end of the first linker portion; further, the DNA encoding the second of the three domains is ligated to one end of the second linker portion, and the DNA encoding the third of the three domains is ligated to the other end of the second linker site, where the first domain, the second domain, and the third domain are different and the first domain, the second domain, and the third domain are independently selected from domain A, domain B, and domain C. Such ligation is performed, for example, sequentially or as a three-way ligation.

[0090] CD3-binding domain The specificity of T cell responses is mediated by the recognition of antigens (presented in the context of the major histocompatibility complex, MHC) by the TCR. As part of the TCR, CD3 is a protein complex that exists on the cell surface and contains the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. To contain the complete TCR, CD3 binds together with the α (alpha) and β (beta) chains of the TCR, as well as CD3ζ (zeta). Clustering CD3 on T cells, such as by using immobilized anti-CD3 antibodies, induces T cell activation that resembles TCR binding but is independent of the specificity typical of its clone.

[0091] In one aspect, the DLL3-targeted trispecific proteins described herein include a domain that specifically binds to CD3. In one aspect, the DLL3-targeted trispecific proteins described herein include a domain that specifically binds to human CD3. In some embodiments, the DLL3-targeted trispecific proteins described herein include a domain that specifically binds to CD3γ. In some embodiments, the DLL3-targeted trispecific proteins described herein include a domain that specifically binds to CD3δ. In some embodiments, the DLL3-targeted trispecific proteins described herein include a domain that specifically binds to CD3ε.

[0092] In further embodiments, the DLL3-targeted trispecific proteins described herein include a domain that specifically binds to the TCR. In certain examples, the DLL3-targeted trispecific proteins described herein include a domain that specifically binds to the α chain of the TCR. In certain examples, the DLL3-targeted trispecific proteins described herein include a domain that specifically binds to the β chain of the TCR.

[0093] In certain embodiments, the CD3 binding domain of the DLL3-targeting trispecific protein described herein not only exhibits strong CD3 binding affinity with human CD3, but also shows excellent cross-reactivity with each cynomolgus CD3 protein.

[0094] In some embodiments, the CD3 binding domain of the DLL3 trispecific antigen-binding protein can be any domain that binds to CD3, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. In some instances, it may be beneficial for the CD3 binding domain to be derived from the same species in which the DLL3 trispecific antigen-binding protein will ultimately be used. For example, for use in humans, it may be beneficial for the CD3 binding domain of the DLL3 trispecific antigen-binding protein to include human or humanized residues from the antigen-binding domain of an antibody or antibody fragment.

[0095] Accordingly, in one aspect, the antigen-binding domain includes a humanized antibody or human antibody or antibody fragment, or a mouse antibody or antibody fragment. In one embodiment, the humanized or human anti-CD3 binding domain includes one or more (e.g., all three) of the light chain complementarity determining regions 1 (LC CDR1), light chain complementarity determining regions 2 (LC CDR2), and light chain complementarity determining regions 3 (LC CDR3) of the humanized or human anti-CD3 binding domain described herein, and / or one or more (e.g., all three) of the heavy chain complementarity determining regions 1 (HC CDR1), heavy chain complementarity determining regions 2 (HC CDR2), and heavy chain complementarity determining regions 3 (HC CDR3) of the humanized or human anti-CD3 binding domain described herein, including one or more, all three LC CDRs and one or more, all three HC CDRs of the humanized or human anti-CD3 binding domain.

[0096] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human light chain variable region specific for CD3, and the light chain variable region specific for CD3 comprises human or non-human light chain CDRs in a human light chain framework region. In one example, the light chain framework region is a λ (lambda) light chain framework. In other examples, the light chain framework region is a κ (kappa) light chain framework.

[0097] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human heavy chain variable region specific for CD3, and the heavy chain variable region specific for CD3 comprises human or non-human heavy chain CDRs in a human heavy chain framework region.

[0098] In certain examples, the complementarity determining regions of the heavy and / or light chains are derived from known anti-CD3 antibodies such as, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4), teprotumumab (MGA031), visilizumab (Nuvion), SP34, 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.

[0099] In one embodiment, the anti-CD3 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 variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the variable regions of the light and heavy chains are continuously linked by a short flexible polypeptide linker and can be expressed as a single polypeptide chain, and the scFv retains the specificity of the intact antibody from which it is derived. In embodiments, the anti-CD3 binding domain comprises: a light chain variable region having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence provided herein, but having at most 30, 20, or 10 modifications (e.g., substitutions), or a light chain variable region comprising a sequence having 95-99% identity to the amino acid sequence provided herein; and / or a heavy chain variable region having at least one, two, or three modifications (e.g., substitutions) of the amino acid sequence provided herein, but having at most 30, 20, or 10 modifications (e.g., substitutions), or a heavy chain variable region comprising a sequence having 95-99% identity to the amino acid sequence provided herein. In some examples, the anti-CD3 binding domain comprises a sequence selected from SEQ ID No. 1793-1807, 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-CD3 binding domain comprises three heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR3) and three light chain CDRs. The heavy chain CDR1 (HC CDR1) of the CD3 binding domain comprises a sequence selected from SEQ ID No. 1820-1831, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID No. 1820-1831, or is at least about 80% to about 99%.The heavy chain CDR2 (HC CDR2) of the CD3 binding domain comprises a sequence selected from SEQ ID No. 1832-1841, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID No. 1832-1841. The heavy chain CDR3 (HC CDR3) of the CD3 binding domain comprises a sequence selected from SEQ ID No. 1842-1853, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID No. 1842-1853. The light chain CDR1 (LC CDR1) of the CD3 binding domain comprises a sequence selected from SEQ ID No. 1852-1864, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID No. 1852-1864. The light chain CDR2 (LC CDR2) of the CD3 binding domain comprises a sequence selected from SEQ ID No. 1865-1877, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID No. 1865-1877. The light chain CDR3 (LC CDR3) of the CD3 binding domain comprises a sequence selected from SEQ ID No. 1878-1884, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID No. 1878-1884. In one embodiment, the humanized or human anti-CD3 binding domain is a scFv, and the light chain variable region comprising the amino acid sequence described herein is linked by a scFv linker to the heavy chain variable region comprising the amino acid sequence described herein. The light chain variable region and the heavy chain variable region of the scFv can be in either 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.

[0100] In some examples, scFvs that bind to CD3 are prepared according to known methods. For example, an scFv molecule can be generated by joining the VH and VL regions together using a flexible polypeptide linker. The scFv molecule includes an scFv linker (e.g., a Ser-Gly linker) with an optimized length and / or amino acid composition. Accordingly, in some embodiments, the length of the scFv linker is such that the VH or VL domain can bind intermolecularly with other variable domains to form the CD3 binding site. In one embodiment, such scFv linkers are "short", i.e., consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in one example, the 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, these scFv linkers consist of about 3 to about 15, such as 8, 9, or 10, contiguous amino acid residues. With regard to the amino acid composition of the scFv linker, a peptide is selected that confers flexibility, does not interfere with the variable domains, and allows for interchain folding to join the two variable domains to form a functional CD3 binding site. For example, an scFv linker containing glycine and serine residues generally confers protease resistance. In some embodiments, the linker in the scFv contains glycine and serine residues. The amino acid sequence of the scFv linker can be optimized, for example, by phage display methods that improve CD3 binding and the production yield of the scFv. Examples of peptide scFv linkers suitable for linking the variable light chain domain and the variable heavy chain domain in an scFv include, but are not limited to, (GS) n (SEQ ID No.1809), (GGS) n (SEQ ID No.1810), (GGGS) n (SEQ ID No.1811), (GGSG) n (SEQ ID No.1812), (GGSGG) n (SEQ ID No.1813), (GGGGS) n(SEQ ID No.1814), (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 can be (GGGGS)4 (SEQ ID NO:1817), or (GGGGS)3 (SEQ ID NO:1818). In some embodiments, the linker comprises an array formed from any combination of the linkers specified in SEQ ID No.1809 - 1818, and the length of such a linker is, in some examples, at most 15 amino acids, or longer than 15 amino acids. Variations in linker length can retain or enhance activity and may result in excellent efficacy in activity studies.

[0101] In some embodiments, the CD3 binding domain of the DLL3 - targeted trispecific antigen - binding protein has an affinity for CD3 on CD3 - expressing cells with a KD of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In some embodiments, the CD3 binding domain of the DLL3 - targeted trispecific antigen - binding protein has an affinity for CD3ε, γ, or δ with a KD of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In a further embodiment, the CD3 binding domain of the DLL3 - targeted trispecific antigen - binding protein has a low affinity for CD3 (i.e., about 100 nM or more).

[0102] The affinity for binding to CD3 can be determined, for example, by the ability of the DLL3-targeted trispecific antigen-binding protein itself or its CD3-binding domain to bind to CD3 coated on an assay plate; displayed on the surface of microbial cells; in solution, etc. The binding activity of the DLL3-targeted trispecific antigen-binding protein itself or its CD3-binding domain of the present disclosure to CD3 can be analyzed by immobilizing a ligand (e.g., CD3), or the DLL3-targeted trispecific antigen-binding protein itself or its CD3-binding domain, onto beads, substrates, cells, etc. The agent can be added to the binding partner cultured in an appropriate buffer at a predetermined temperature for a certain period. After washing to remove unbound material, the bound protein can be released, for example, with SDS, high pH buffer, etc., and can be analyzed, for example, by surface plasmon resonance (SPR).

[0103] Half-life extension domain As used herein, domains that extend the half-life of an antigen-binding domain are contemplated. Such domains are contemplated to include, but are not limited to, albumin-binding domains, Fc domains, small molecules, and other half-life extension domains known in the art.

[0104] Human albumin (ALB) (molecular mass of 67 kDa) is the most abundant protein in plasma, present at about 50 mg / ml (600 μM), and has a half-life of about 20 days in humans. ALB helps to maintain plasma pH, contributes to the colloidal osmotic pressure, functions as a carrier for many metabolites and fatty acids, and serves as a major drug transport protein in plasma.

[0105] Non-covalent association with albumin extends the elimination half-life of short-lived proteins. For example, recombinant fusion of an albumin-binding domain to a Fab fragment resulted in 25-fold and 58-fold in vivo clearance and 26-fold and 37-fold half-life extension upon intravenous administration to mice and rabbits, respectively, compared to administration of the Fab fragment alone. In another example, when insulin was acylated with fatty acids to promote association with albumin, a delayed effect was observed when injected subcutaneously into rabbits or pigs. Overall, such studies demonstrate the linkage between albumin binding and delayed action.

[0106] In one aspect, the DLL3-targeted trispecific proteins described herein include a half-life extension domain, e.g., a domain that specifically binds to ALB. In some embodiments, the ALB-binding domain of the DLL3-targeted trispecific antigen-binding protein can be any domain that binds to ALB, including but not limited to domains derived 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) specific for HSA, a single-domain antibody, e.g., a variable heavy domain (VH), a variable light domain (VL), and a variable domain of a single-domain antibody derived from camelids (VHH), a peptide, a ligand, or a small molecule entity. In one embodiment, 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. The HSA-binding domain of the DLL3 trispecific antigen-binding protein is quite small and is contemplated to be 25 kDa or less, 20 kDa or less, 15 kDa or less, or 10 kDa or less in some embodiments. In one example, the ALB binding is 5 kDa or less when it is a peptide or a small molecule entity.

[0107] The half-life extension domain of the DLL3-targeted trispecific antigen-binding protein results in changes in the pharmacokinetics and pharmacodynamics of the DLL3-targeted trispecific antigen-binding protein itself. As described above, the half-life extension domain extends the elimination half-life. The half-life extension domain also alters the pharmacokinetic properties, including changes in the tissue distribution, penetration, and diffusion of the trispecific antigen-binding protein. In some embodiments, the half-life extension domain results in improved tissue (including tumor) targeting, tissue distribution, tissue penetration, diffusion within the tissue, and enhanced efficacy as compared to a protein without the half-life extension domain. In one embodiment, the treatment method effectively and efficiently utilizes a reduced amount of the trispecific antigen-binding protein, resulting in a reduction in side effects such as a decrease in the cytotoxicity of non-tumor cells.

[0108] Furthermore, the binding affinity of the half-life extension domain can be selected to target the specific elimination half-life of a particular trispecific antigen-binding protein. Thus, in some embodiments, the half-life extension domain has a high binding affinity. In other embodiments, the half-life extension domain has a moderate binding affinity. In still other embodiments, the half-life extension domain has a low or weak binding affinity. Typical binding affinities include KD concentrations of less than 10 nM (high), between 10 nM and 100 nM (moderate), and greater than 100 nM (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 domain described herein comprises a single-domain antibody.

[0109] In some embodiments, the half-life extension domain is a sequence selected from SEQ ID No. 1769-1778, or a sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to a sequence selected from SEQ ID No. 1769-1778. In some examples, the half-life extension comprises three heavy-chain CDRs (HC CDR1, HC CDR2, and HC CDR3) and three light-chain CDRs. In some examples, the half-life extension comprises three heavy-chain CDRs (HC CDR1, HC CDR2, and HC CDR3) or three light-chain CDRs. In some embodiments, the heavy-chain CDR1 (HC CDR1) of the half-life extension domain is a sequence selected from SEQ ID No. 1782-1784, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID No. 1782-1784, or comprising at least about 80% to about 99%. In some embodiments, the heavy-chain CDR2 (HC CDR2) of the half-life extension domain comprises a sequence selected from SEQ ID No. 1785-1790, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID No. 1785-1790. The heavy-chain CDR3 (HC CDR3) of the CD3 binding domain comprises a sequence selected from SEQ ID No. 1791 or 1792, or a sequence comprising one or more modifications or substitutions of a sequence selected from SEQ ID No. 1791 or 1792.

[0110] DLL3 binding domain DLL3 (also known as delta-like ligand 3 or SCDO1) is a member of the delta-like family of Notch DSL ligands. Representative DLL3 protein orthologs include, but are not limited to, human (accession numbers NP_058637 and NP_982353), chimpanzee (accession number XP_003316395), mouse (accession number NP_031892), and rat (accession number NP_446118). In humans, the DLL3 gene consists of 8 exons spanning 9.5 kbp on chromosome 19q13. Alternative splicing within the last exon results in one of two processed transcripts, one of 2389 bases (accession number NM_016941) and one of 2052 bases (accession number NM_203486). The former transcript encodes a 618 amino acid protein (accession number NP_058637), while the latter encodes a 587 amino acid protein (accession number NP_982353). These two protein isoforms of DLL3 share overall 100% identity across their extracellular domains and their transmembrane domains, and differ only in that the longer isoform contains an extended cytoplasmic tail with 32 additional residues at the carboxy terminus of the protein. The extracellular region of the DLL3 protein contains 6 EGF-like domains, a single DSL domain, and an N-terminal domain. In general, the EGF domains occur at amino acid residues approximately 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), the DSL domain occurs at amino acid residues approximately 176 - 215, and the N-terminal domain occurs at amino acid residues approximately 27 - 175 of hDLL3. Each of the EGF-like domain, DSL domain, and N-terminal domain contains a portion of the DLL3 protein defined by different amino acid sequences. The EGF-like domains are, in some embodiments, referred to as EGF6 to EGF1, with EGF1 being closer to the N-terminal group portion of the protein.Generally, DSL ligands consist of the following series of structural domains: a unique N-terminal domain, followed by a conserved DSL domain, a number of tandem epidermal growth factor (EGF)-like repeats, a transmembrane domain, and a cytoplasmic domain that contains multiple lysine residues that are not highly conserved across ligands but are potential sites for ubiquitination by a unique E3 ubiquitin ligase. The DSL domain is a degenerate EGF domain that is necessary but not sufficient for interaction with the Notch receptor. Furthermore, the first two EGF-like repeats of most DSL ligands contain a smaller protein sequence motif known as the DOS domain that interacts cooperatively with the DSL domain when activating Notch signaling.

[0111] In some embodiments, the disclosed DLL3 trispecific binding proteins of this disclosure are generated, made, engineered, or selected to react with selected domains, motifs, or epitopes within the DLL3 protein. In some embodiments, the DLL3-targeted trispecific protein binds to the DSL domain, and in some embodiments, binds to an epitope that includes G203, R205, P206 within the DSL domain.

[0112] The DLL3 binding domain of the DLL3-targeted trispecific proteins of this disclosure is engineered, made, and / or selected to react with both isoforms of DLL3 or a single isoform of the protein in some embodiments, or, conversely, includes a pan-DLL binding domain that reacts with or associates with at least one additional DLL family member in addition to DLL3. In some embodiments, a DLL3 binding domain, such as the DLL3 binding domain, is engineered, made, and / or selected to react with a domain (or an epitope therein) represented only by DLL3 or with a domain that is conserved at least somewhat across multiple or all DLL family members.

[0113] In some embodiments, the DLL3 binding domain associates or binds to a specific epitope, moiety, motif, or domain of DLL3. Both DLL3 isoforms incorporate the same extracellular region, including at least an N-terminal domain, a DSL (Delta / Serrate / lag-2) domain, and six EGF-like domains (i.e., EGF1-EGF6). Thus, in one embodiment, the DLL3 binding domain binds 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 binds or 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 to a specific epitope in a particular EGF-like domain of DLL3. In some embodiments, the DLL3 binding domain associates or binds to an epitope in EGF1 (amino acids 216-249), EGF2 (amino acids 274-310), EGF3 (amino acids 312-351), EGF4 (amino acids 353-389), EGF5 (amino acids 391-427), or EGF6 (amino acids 429-465). In some embodiments, each of the aforementioned domains includes one or more epitopes and / or one or more bins. 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 an epitope therein. In some embodiments, the DLL3 binding domain binds, reacts with, or associates with the N-terminal domain or an epitope therein.

[0114] In some embodiments, the DLL3-binding proteins of the present disclosure, such as the DLL3-binding domain of the trispecific proteins of the present disclosure, bind to the full-length DLL3 protein or a fragment thereof, such as an epitope-containing fragment within the full-length DLL3 protein, as described above. In some cases, the epitope-containing fragment includes antigenic or immunogenic fragments of the DLL3 protein and derivatives thereof. The epitope-containing fragment containing the antigenic or immunogenic fragment is, in some embodiments, 12 or more amino acids, 20 or more amino acids, 50 or more, or 100 amino acids. In some embodiments, the DLL3 fragment comprises 95% or more, 90%, or 75% or 50% or 25% or 10% or more of the length of the full protein. In some embodiments, the epitope-containing fragment of DLL3 containing the antigenic or immunogenic fragment can induce a relevant immune response in a patient. The derivatives of DLL3, in some embodiments, have 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% depending on the number of amino acids based on the full length of the protein) deletions, insertions, or substitutions with respect to the DLL3 sequence provided by SEQ ID No. 1885 (UniProtKB Accession Q9NYJ7). In some embodiments, the substitutions include conservative substitutions. The derivatives and variants of DLL3, in some examples, have essentially the same biological function as the DLL3 protein from which they are derived. For example, the derivatives and variants of DLL3 are, in some cases, relatively antigenic or immunogenic with respect to the protein from which they are derived, and have either the ligand-binding activity of the protein from which they are derived, or the ability to form an active receptor complex, or preferably both, and have the same tissue distribution as DLL3.

[0115] By designing the DLL3-targeting trispecific protein described in this specification, the binding domain for DLL3 can be made flexible in that it can be any type of binding domain that includes, but is not limited to, domains derived from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the binding domain for DLL3 is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy-chain variable domain (VH), a light-chain variable domain (VL), and a variable domain of a single-domain antibody derived from camel (VHH). In other embodiments, the binding domain for DLL3 is a non-Ig binding domain, i.e., an antibody mimetic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, knotted domain peptides, and monobodies. In further embodiments, the binding domain for DLL3 is a ligand or peptide that binds or associates with DLL3. In further embodiments, the binding domain for DLL3 is a notchtin. In further embodiments, the binding domain for DLL3 is a small molecule entity.

[0116] In some embodiments, the DLL3 binding domain binds to a protein comprising the sequence of SEQ ID NO:1885 (UniProtKB Accession Q9NYJ7). In some embodiments, the DLL3 binding domain binds to a protein comprising a truncated sequence as compared to SEQ ID NO:1885 (UniProtKB Accession Q9NYJ7). In some embodiments, the DLL3 binding domain binds to a protein comprising the sequence of SEQ ID No.1892 or SEQ ID No.1893 (the mature extracellular domain of the DLL3 protein). In some embodiments, the DLL3 binding domain binds to a protein comprising amino acids 47 - 492 of SEQ ID No.1892. In some embodiments, the DLL3 binding domain recognizes an epitope within amino acids 47 - 4492 of SEQ ID No.1892.

[0117] In some embodiments, the DLL3 binding domain is an anti-DLL3 antibody or antibody variant. As used herein, the term "antibody variant" refers to variants and derivatives of the antibodies described herein. In certain embodiments, amino acid sequence variants of the anti-DLL3 antibodies described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-DLL3 antibodies described herein are contemplated to improve the binding affinity and / or other biological properties of the antibody. Exemplary methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or peptide synthesis. Such modifications include, for example, deletions from, and / or insertions and / or substitutions of, residues within the amino acid sequence of the antibody.

[0118] To obtain the final construct, any combination of deletions, insertions, and substitutions can be made, provided that the final construct has antigen binding, which is the desired feature. In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introduced into the antibody of interest, and the products may be screened for the desired activity, retained / improved antigen binding, reduced immunogenicity, or improved T cell-mediated cytotoxicity (TDCC). Both conservative and non-conservative amino acid substitutions are contemplated for the preparation of antibody variants.

[0119] In another example of substitution for generating a variant anti-DLL3 antibody, one or more residues of the hypervariable regions of the parental antibody are substituted. Generally, variants are selected based on improved desired properties compared to the parental antibody, such as increased affinity, decreased affinity, decreased immunogenicity, increased pH-dependence of binding.

[0120] In some embodiments, the DLL3-binding domain of the DLL3-targeting trispecific protein is a single-domain antibody specific for DLL3, such as a variable domain of a heavy chain (VH), a variable domain of a single-domain antibody from llama (VHH), a peptide, a ligand, or a small molecule entity. In some embodiments, the DLL3-binding domain of the DLL3-targeting trispecific protein described herein is any domain that binds to DLL3, including but not limited to 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.

[0121] In general, it should be noted that the term single-domain antibody, as used herein in its broadest sense, is not limited to a particular biological source or a particular preparation method. A single-domain antibody is an antibody in which its complementarity-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies lacking a light chain by nature, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. A single-domain antibody can be any antibody in the art or any future single-domain antibody. A single-domain antibody can be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and cow. For example, in some embodiments, the single-domain antibodies of the present disclosure are obtained by (1) isolation of the VHH domain of a naturally occurring heavy-chain antibody; (2) expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) "humanization" of a naturally occurring VHH domain or expression of a nucleic acid encoding such a humanized VHH domain; (4) "camelization" of a naturally occurring VH domain derived from any animal species, and particularly from mammalian species such as humans, or expression of a nucleic acid encoding such a camelized VH domain; (5) "camelization" of a "domain antibody" or "Dab", or expression of a nucleic acid encoding such a camelized VH domain; (6) use of synthetic or semi-synthetic techniques for preparing proteins, polypeptides, or other amino acid sequences; (7) preparation of a nucleic acid encoding a single-domain antibody using nucleic acid synthesis techniques known in the art, followed by expression of the nucleic acid thus obtained; and / or (8) obtained by any combination of one or more of the foregoing.

[0122] In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody directed against DLL3. As further described herein, such VHH sequences are generally generated or obtained by appropriately immunizing a camelid species having DLL3 (i.e., to produce an immune response and / or heavy chain antibodies directed against DLL3), obtaining an appropriate biological sample (such as a blood sample, a serum sample, or a sample of B cells) from said camelid, and generating VHH sequences directed against DLL3 starting from said sample using any suitable technique known in the art.

[0123] In another embodiment, such a naturally occurring VHH domain against DLL3 is obtained from a naïve library of VHH sequences of camelids by screening such a library using, for example, at least one screening technique known in the art, using DLL3, or at least one portion, fragment, epitope, or antigenic determinant thereof. Such libraries and techniques are described, for example, in WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. Alternatively, an improved synthetic or semi-synthetic library derived from a naïve VHH library, such as a VHH library obtained from a naïve VHH library by techniques such as random mutagenesis and / or CDR shuffling, as described in WO00 / 43507, is used.

[0124] In a further embodiment, additional techniques for obtaining VHH sequences directed against DLL3 include appropriately immunizing transgenic mammals capable of expressing heavy chain antibodies (i.e., for generating an immune response and / or producing heavy chain antibodies directed against DLL3), obtaining an appropriate biological sample (blood sample, serum sample, or sample of B cells) from the transgenic mammal, and generating VHH sequences directed against DLL3 starting from the sample using any suitable technique known in the art. For example, for this purpose, heavy chain antibody-expressing rats or mice, and additional methods and techniques described in WO02 / 085945 and WO04 / 049794 can be used.

[0125] In some embodiments, the anti-DLL3 single-domain antibodies of the DLL3-targeting trispecific proteins correspond to the amino acid sequences of naturally occurring VHH domains but are "humanized", i.e., one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (and, in particular, within the framework sequence) are replaced by one or more of the amino acid residues occurring at the corresponding positions in the VH domain from a conventional 4-chain antibody of human origin (such as those shown above), and include single-domain antibodies having an amino acid sequence that is "humanized". This can be carried out in a manner known in the art that will be apparent to those skilled in the art based on, for example, further description herein. Further, such humanized anti-DLL3 single-domain antibodies of the present disclosure are obtained in any suitable manner known per se (i.e., as shown in items (1) to (8) above), and thus are not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as a starting material. In some further embodiments, the single-domain anti-DLL3 antibodies correspond to the amino acid sequences of naturally occurring VH domains as described herein but are "camelized", i.e., one or more amino acid residues in the amino acid sequence of the naturally occurring VH domain from a conventional 4-chain antibody are replaced by one or more of the amino acid residues occurring at the corresponding positions in the VHH domain of a heavy-chain antibody, and include single-domain antibodies having an amino acid sequence that is "camelized". Such "camelization" substitutions preferably occur at the VH-VL interface and / or form residues prominent in so-called camelids, and / or are inserted at amino acid positions present at the VH-VL interface and / or in residues prominent in so-called camelids (see, for example, WO94 / 04678 and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence used as a starting material or starting point for generating or designing the camelized single domain is preferably a VH sequence derived from a mammal, more preferably a human VH sequence such as the VH3 sequence.However, in certain embodiments, such camelized anti-DLL3 single-domain antibodies of the present disclosure are obtained in a manner known in the art (i.e., as shown in items (1)-(8) above), and thus it should be noted that they are not strictly limited to polypeptides obtained using a naturally occurring VH domain as a starting material. For example, as further described herein, both "humanization" and "camelization" involve providing a naturally occurring VHH domain or a nucleotide sequence encoding a VH domain, and then changing one or more codons in the nucleotide sequence so that the new nucleotide sequence encodes a "humanized" or "camelized" single-domain antibody, respectively. This nucleic acid is then expressed, and as a result, the desired anti-DLL3 single-domain antibody of the present disclosure can be obtained. Alternatively, in other embodiments, the amino acid sequences of the desired humanized or camelized anti-DLL3 single-domain antibodies of the present disclosure are designed based on the amino acid sequences of the naturally occurring VHH domain or VH domain, respectively, and then de novo synthesized using known techniques of peptide synthesis. In some embodiments, nucleotide sequences encoding the desired humanized or camelized anti-DLL3 single-chain antibodies of the present disclosure are designed based on the amino acid sequences or nucleotide sequences of the naturally occurring VHH domain or VH domain, respectively, then de novo synthesized using known techniques of nucleic acid synthesis, and then the thus obtained nucleic acid is expressed using known expression techniques, and as a result, the desired anti-DLL3 single-domain antibody of the present disclosure is obtained.

[0126] Other suitable methods and techniques for obtaining the anti-DLL3 single-domain antibodies of the present disclosure and / or nucleic acids encoding the same starting from naturally occurring VH or VHH sequences 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 semi-synthetic sequences in a suitable manner to provide the anti-DLL3 single-domain antibodies of the present disclosure or nucleotide sequences or nucleic acids encoding the same.

[0127] In some embodiments, the DLL3-binding domain is an anti-DLL3 specific antibody comprising heavy chain variable complementarity-determining region CDR1, heavy chain variable CDR2, heavy chain variable CDR3, light chain variable CDR1, light chain variable CDR2, and light chain variable CDR3. In some embodiments, the DLL3-binding domain includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, or antigen-binding fragments, such as single-domain antibodies (sdAb), Fab, Fab’, F(ab)2, and Fv fragments, fragments composed of one or more CDRs, single-chain antibodies (such as single-chain Fv fragments (scFv)), disulfide-stabilized (dsFv) Fv fragments, heteroconjugate antibodies (such as bispecific antibodies), pFv fragments, heavy chain monomers or heavy chain dimers, light chain monomers or light chain dimers, and domains from dimers consisting of one heavy chain and one light chain, and any domain that binds to DLL3. In some embodiments, the DLL3-binding domain is a single-domain antibody. In some embodiments, the anti-DLL3 single-domain antibody comprises heavy chain variable complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3.

[0128] In some embodiments, the DLL3 binding domain is a polypeptide comprising an amino acid sequence composed of four framework regions / sequences (f1-f4) interrupted by three complementarity determining regions / sequences, as represented by the following formula: f1-r1-f2-r2-f3-r3-f4, where r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues. The framework residues of the DLL3 binding protein of the present disclosure include, for example, amino acid residues 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94, and the complementarity determining regions include, for example, amino acid residues 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36. In some embodiments, the DLL3 binding domain comprises an amino acid sequence selected from SEQ ID NO: 1-442 and 1886. In some embodiments, CDR1 of the DLL3 binding domain comprises a sequence selected from SEQ ID No. 443-884 and 1887, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID No. 443-884 and 1887. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID No. 885-1326 and 1888, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID No. 885-1326 and 1888. In some embodiments, CDR3 comprises a sequence selected from SEQ ID No. 1327-1768 and 1889, or one or more substitutions relative to a sequence selected from the group consisting of SEQ ID No. 1327-1768 and 1889.

[0129] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID No. 443-884 and 1887, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the amino acids selected from SEQ ID No. 443-884 and 1887. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID No. 885-1326 and 1888, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the amino acid sequence selected from SEQ ID No. 885-1326 and 1888. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID No. 1327-1768 and 1889, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the sequence selected from SEQ ID No. 1327-1768 and 1889.

[0130] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID No. 495-528, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the amino acids selected from SEQ ID No. 495-528. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID No. 937-970, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the amino acid sequence selected from SEQ ID No. 937-970. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID No. 1379-1412, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the sequence selected from SEQ ID No, 1379-1412.

[0131] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID No. 529-809, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the amino acids selected from SEQ ID No. 529-809. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID No. 971-1251, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the amino acid sequence selected from SEQ ID No. 971-1251. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID No. 1379-1412, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the sequence selected from SEQ ID No. 1379-1412.

[0132] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID No. 810-884, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the amino acids selected from SEQ ID No. 810-884. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID No. 1252-1326, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the amino acid sequence selected from SEQ ID No. 1252-1326. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID No. 1692-1768, or a variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions in the sequence selected from SEQ ID No. 1692-1768.

[0133] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID No. 1-442 and 1886. In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID No. 53-86.

[0134] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NO. 87-367.

[0135] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO. 68, or a sequence derived from SEQ ID NO. 68.

[0136] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO. 75, or a sequence derived from SEQ ID NO. 75.

[0137] In some embodiments, the DLL3 binding domain of the DLL3-targeting trispecific binding protein is cross-reactive with human and cynomolgus DLL3. In some embodiments, the DLL3 binding domain is specific for human DLL3. In certain embodiments, the DLL3 binding domains disclosed herein bind to human DLL3 with a human Kd (hKd). In certain embodiments, the DLL3 binding domains disclosed herein bind to cynomolgus DLL3 with a cynomolgus Kd (cKd). In certain embodiments, the DLL3 binding domains disclosed herein bind to both cynomolgus DLL3 and human DLL3 (hKd), each having a cynomolgus Kd (cKd) and a human Kd, respectively. In some embodiments, the DLL3 binding protein binds to human and cynomolgus DLL3 with equivalent binding affinities (i.e., the values of hKd and cKd do not differ by more than ±10%). In some embodiments, hKd and cKd are in the range of about 0.001 nM to about 500 nM. In some embodiments, hKd and cKd are in the range of about 0.001 nM to about 450 nM. In some embodiments, hKd and cKd are in the range of about 0.001 nM to about 400 nM. In some embodiments, hKd and cKd are in the range of about 0.001 nM to about 350 nM. In some embodiments, hKd and cKd are in the range of about 0.001 nM to about 300 nM. In some embodiments, hKd and cKd are in the range of about 0.001 nM to about 250 nM. In some embodiments, hKd and cKd are in the range of about 0.001 nM to about 200 nM. In some embodiments, hKd and cKd are in the range of about 0.001 nM to about 150 nM. In some embodiments, hKd and cKd are in the range of about 0.001 nM to about 100 nM. In some embodiments, hKd and cKd are in the range of about 0.1 nM to about 90 nM. In some embodiments, hKd and cKd are in the range of about 0.2 nM to about 80 nM. In some embodiments, hKd and cKd are in the range of about 0.3 nM to about 70 nM. In some embodiments, hKd and cKd are in the range of about 0.4 nM to about 50 nM. In some embodiments, hKd and cKd are in the range of about 0.5 nM to about 30 nM.In some embodiments, hKd and cKd are in the range of about 0.6 nM to about 10 nM. In some embodiments, hKd and cKd are in the range of about 0.7 nM to about 8 nM. In some embodiments, hKd and cKd are in the range of about 0.8 nM to about 6 nM. In some embodiments, hKd and cKd are in the range of about 0.9 nM to about 4 nM. In some embodiments, hKd and cKd are in the range of about 1 nM to about 2 nM.

[0138] 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 cells that express DLL3. Soluble DLL3 refers to DLL3 that is expressed or that is no longer present in or on the cell membrane surface of cells that expressed DLL3. In one example, soluble DLL3 is present in the blood and / or lymph circulation of a subject. In one embodiment, the DLL3 binding protein binds 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 more to membrane-bound DLL3 compared to soluble DLL3. In one embodiment, the trispecific antigen-binding protein of the present disclosure preferentially binds 30-fold more to membrane-bound DLL3 compared to soluble DLL3. The determination of preferential binding of an antigen-binding protein to membrane-bound DLL3 over soluble DLL3 can be readily determined using assays well known in the art.

[0139] In some embodiments, any of the foregoing DLL3 binding domains (e.g., anti-DLL3 single domain antibodies of SEQ ID NOs: 1-442 and 1886) are affinity peptides tagged for ease of purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, called 6X-his (SEQ ID No. 1819).

[0140] In some embodiments, any of the aforementioned DLL3 binding domains (e.g., anti-DLL3 single domain antibodies of SEQ ID No. 1-442 and 1886) is an affinity peptide tagged for easy purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, called 6X-his (SEQ ID No. 1819).

[0141] Integration into Chimeric Antigen Receptor (CAR) The DLL3-targeted trispecific antigen-binding proteins of the present disclosure can, in some examples, be incorporated into a chimeric antigen receptor (CAR). Engineered immune effector cells, T cells, or NK cells can be used to express a CAR comprising an anti-DLL3-targeted trispecific protein containing an anti-DLL3 single domain antibody as described herein. In one embodiment, a CAR comprising an anti-DLL3-targeted trispecific protein described herein is linked via a hinge region to a transmembrane domain and further to a co-stimulatory domain, OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or a functional signaling domain obtained from 4-1BB. In some embodiments, the CAR further comprises a sequence encoding an intracellular signaling domain such as 4-1BB and / or CD3 zeta.

[0142] Tumor Growth Inhibitory Properties In certain embodiments, the DLL3-targeted trispecific proteins of the present disclosure, when administered to a subject having tumor cells that express DLL3, decrease the growth of tumor cells in vivo. The decrease in tumor cell growth can be determined by a variety of methods well known in the art. Non-limiting examples include direct measurement of tumor dimensions, measurement of the amount of excised tumor and comparison to control subjects, and measurement by imaging techniques (e.g., CT or MRI) that may or may not use isotopes or luminescent molecules (e.g., luciferase) to enhance the analysis.

[0143] In certain embodiments, administration of the trispecific proteins of the present disclosure results in at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the in vivo growth of tumor cells compared to a control antigen-binding agent, and a reduction of about 100% in tumor growth indicates complete remission and disappearance of the tumor. In further embodiments, administration of the trispecific proteins of the present disclosure results in a reduction of about 50-100%, about 75-100%, or about 90-100% in the in vivo growth of tumor cells compared to a control antigen-binding agent. In further embodiments, administration of the trispecific proteins of the present disclosure results in a reduction of about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100% in the in vivo growth of tumor cells compared to a control antigen-binding agent.

[0144] Modifications of DLL3-targeted trispecific proteins The DLL3-targeted trispecific proteins described herein include derivatives or analogs in which (i) an amino acid is substituted with an amino acid residue not encoded by the genetic code, (ii) the mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein such as a leader or secretory sequence, or a sequence for purification of the protein.

[0145] Typical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, tRNA-mediated addition of amino acids to proteins, e.g., arginylation, and ubiquitination.

[0146] Modifications are made at any location of the DLL3-targeted trispecific proteins described herein, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Specific general peptide modifications useful for modification of the DLL3-targeted trispecific proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamate residues, hydroxylation, blocking of amino or carboxyl groups, or both, in the polypeptide by covalent modification, and ADP-ribosylation.

[0147] In some embodiments, derivatives of the DLL3-targeted trispecific proteins described herein include derivatives of immunoreactive modulators and antigen-binding molecules that include one or more modifications.

[0148] In some embodiments, the trispecific DLL3 binding molecules of the present disclosure are monovalent or multivalent, such as bivalent, trivalent, etc. As used herein, the term "valence" refers to the number of potential target binding sites that associate with an antibody. Each target binding site specifically binds to one target molecule or a specific location or locus on the target molecule. When an antibody is monovalent, each binding site of the molecule specifically binds to a single antigenic position or epitope. When an antibody contains more than one target binding site (is multivalent), each target binding site can specifically bind to the same or different molecules (e.g., can bind to different ligands or different antigens, or different epitopes or positions on the same antigen).

[0149] In some embodiments, the DLL3-targeted trispecific proteins of the present disclosure include, inter alia, one or more additional amino acid residue substitutions, mutations, and / or modifications, which result in compounds having, but not limited to, the following preferred properties: altered pharmacokinetics, increased serum half-life, increased binding affinity, decreased immunogenicity, increased production, changes in the Fc ligand that binds to the Fc receptor (FcR), enhancement or reduction of the activity of "ADCC" (antibody-dependent cellular cytotoxicity) or "CDC" (complement-dependent cytotoxicity), changes in glycosylation and / or disulfide bonds, and modification of binding specificity. In some cases, these DLL3-targeted trispecific protein variants are advantageously used to enhance the effective antitumor properties of the disclosed DLL3-targeted trispecific proteins.

[0150] In some embodiments, the DLL3-targeted trispecific protein of the present disclosure has a half-life in mammals such as humans or cynomolgus monkeys of less than about 5 days, about 5 days, longer than about 5 days, longer than 10 days, longer than about 15 days, longer than about 20 days, longer than about 25 days, longer than about 30 days, longer than about 35 days, longer than about 40 days, longer than about 45 days, longer than about 2 months, longer than about 3 months, longer than about 4 months, or longer than about 5 months. The increased half-life may in some cases result in a higher serum titer and thus decrease the frequency of administration of the DLL3-targeted trispecific protein, decrease the concentration of the antibody administered, or both.

[0151] Still other embodiments include a DLL3-targeted trispecific binding protein comprising one or more engineered glycoforms, i.e., an altered glycosylation pattern or an altered carbohydrate composition covalently attached to the protein. Engineered glycoforms are useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function, increasing the affinity of the trispecific protein for a target, or promoting the production of the trispecific protein. In some embodiments where a reduction in effector function is desired, the molecule is engineered to express an aglycosylated form. Substitutions that result in the removal of glycosylation sites in one or more variable region frameworks, thereby removing glycosylation at that site, are included in some embodiments. Conversely, in some cases, enhancement of effector function or improvement of binding is imparted to the Fc containing the trispecific protein of this disclosure by engineering at one or more additional glycosylation sites.

[0152] The DLL3-targeted trispecific protein may, in some cases, be differently modified during or after production by, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, binding to antibody molecules or other cellular ligands, etc. Any of a number of chemical modifications may be carried out by techniques including, but not limited to, specific chemical cleavages such as cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formyl group substitution, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

[0153] Various post-translational modifications also included in the present disclosure include, for example, N-linked or O-linked sugar chains, processing of the N-terminal or C-terminal ends, attachment of chemical moieties to the amino acid backbone, chemical modification of N-linked or O-linked sugar chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. Furthermore, the DLL3-targeted trispecific binding protein may, in some cases, be modified with a detectable label, such as an enzymatic, fluorescent, radioisotopic, or affinity label, to enable detection and isolation of the modulator.

[0154] Polynucleotide encoding a DLL3-targeted trispecific protein In some embodiments, polynucleotide molecules encoding the anti-DLL3 trispecific binding proteins described herein are also provided. In some embodiments, the polynucleotide molecules are provided as DNA constructs. In other embodiments, the polynucleotide molecules are provided as messenger RNA transcripts.

[0155] The polynucleotide molecule is constructed by known methods, such as by combining a gene encoding three binding domains separated by a peptide linker or, in other embodiments, directly linked by a peptide bond, into one gene construct operably linked to a suitable promoter and optionally a suitable transcription terminator, and by expressing it in bacteria or other suitable expression systems, such as CHO cells, etc. In embodiments where the DLL3 binding domain is a small molecule, the polynucleotide contains genes encoding the CD3 binding domain and the half-life extension domain. In embodiments where the half-life extension domain is a small molecule, the polynucleotide contains genes encoding the domains that bind to CD3 and DLL3. Depending on the vector system and host utilized, any number of suitable transcriptional and translational elements, including constitutive and inducible promoters, may be used. The promoter is selected to promote the expression of the polynucleotide in each host cell.

[0156] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment. This recombinant vector can be constructed according to known methods. Specific vectors of interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, lentiviruses, etc.), and cosmids.

[0157] Various expression vector / host systems contain the polynucleotide encoding the polypeptide of the trispecific antigen-binding protein described and can be utilized to express this polynucleotide. Examples of expression vectors for expression in E. coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27), or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0158] Accordingly, in some embodiments, the DLL3-targeting trispecific proteins described herein can be produced, isolated, and optionally further purified by introducing a vector encoding the above-described proteins into a host cell and culturing the host cell under conditions that allow expression of the protein domains.

[0159] Pharmaceutical composition In some embodiments, there is also provided a pharmaceutical composition comprising an anti-DLL3 trispecific binding protein described herein, a vector comprising a polynucleotide encoding a polypeptide of a DLL3-targeting trispecific protein, or a host cell transformed with this vector, and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, carriers that do not interfere with the biological activity of the components and are not toxic to the patient to whom they are administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and administered to a subject in appropriate doses. Preferably, the composition is sterilized. These compositions may also contain adjuvants such as preservatives, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents. Further embodiments provide one or more of the above-described DLL3 trispecific proteins packaged in lyophilized form or packaged in an aqueous medium.

[0160] In some embodiments of the pharmaceutical composition, the DLL3 trispecific targeting protein described herein is encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposomes, quantum dots, superparamagnetic microparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical composition, the DLL3-targeted trispecific protein is bound to liposomes. In some examples, the DLL3-targeted trispecific protein is bound to the surface of the liposome. In some examples, the DLL3 trispecific antigen-binding protein is encapsulated within the shell of the liposome. In some examples, the liposome is a cationic liposome.

[0161] The DLL3-targeted trispecific protein described herein is contemplated for use as a medicament. Administration is achieved by different methods, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of treatment and the type of compound included in the pharmaceutical composition. The dosing regimen is determined by the attending physician and other clinical factors. The dosage for any one patient depends on many factors, including the patient's size, body surface area, age, gender, the particular compound being administered, the time and route of administration, the type of treatment, health status, and other drugs being administered concurrently. An "effective amount" refers to an amount of the active ingredient sufficient to affect the course and severity of the disease and thereby cause a reduction or alleviation of such a medical condition, and can be determined using known methods.

[0162] In some embodiments, the DLL3-targeted trispecific protein of the present disclosure is administered at a frequency of once a week at a dose of up to 10 mg / kg. In some cases, the dose is from about 1 ng / kg to about 10 mg / kg. In some embodiments, the dose is from about 1 ng / kg to about 10 ng / kg, about 5 ng / kg to about 15 ng / kg, about 12 ng / kg to about 20 ng / kg, about 18 ng / kg to about 30 ng / kg, about 25 ng / kg to about 50 ng / kg, about 35 ng / kg to about 60 ng / kg, about 45 ng / kg to about 70 ng / kg, about 65 ng / kg to about 85 ng / kg, about 80 ng / kg to about 1 μg / kg, about 0.5 μg / kg to about 5 μg / kg, about 2 μg / kg to about 10 μg / kg, about 7 μg / kg to about 15 μg / kg, about 12 μg / kg to about 25 μg / kg, about 20 μg / kg to about 50 μg / kg, about 35 μg / kg to about 70 μg / kg, about 45 μg / kg to about 80 μg / kg, about 65 μg / kg to about 90 μg / kg, about 85 μg / kg to about 0.1 mg / kg, about 0.095 mg / kg to about 10 mg / kg. In some cases, the dose is from about 0.1 mg / kg to about 0.2 mg / kg; about 0.25 mg / kg to about 0.5 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.75 mg / kg to about 3 mg / kg, about 2.5 mg / kg to about 4 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 6 mg / kg, about 5.5 mg / kg to about 7 mg / kg, about 6.5 mg / kg to about 8 mg / kg, about 7.5 mg / kg to about 9 mg / kg, or about 8.5 mg to 10 mg / kg. The frequency of administration is, in some embodiments, less than approximately daily administration, every other day, less than once a day, twice a week, weekly, once every 7 days, once every 2 weeks, once every 2 weeks, once every 3 weeks, once every 4 weeks, or once a month. In some cases, the frequency of administration is weekly. In some cases, the frequency of administration is weekly and the dose is up to 10 mg / kg. In some cases, the duration of administration is from about 1 day to about 4 weeks or more.

[0163] Treatment method In some embodiments, the DLL3 binding protein, or DLL3-targeting trispecific protein of the present disclosure is administered to treat a neoplastic disease. In some embodiments, the neoplastic disease is benign or malignant; a solid tumor or other blood neoplasia, and in some embodiments, is selected from the group including, but not limited to: adrenal tumors, AIDS-related cancers, alveolar soft part sarcomas, astrocytic tumors, autonomic ganglia tumors, bladder cancer (squamous cell carcinoma and transitional cell carcinoma), blastocoelic disorders, bone cancer (ameloblastoma, aneurysmal bone cyst, osteochondroma, osteosarcoma), cerebrospinal cancer, metastatic brain tumors, breast cancer including triple negative breast cancer, carotid body tumors, cervical cancer, chondrosarcoma, chordoma, chromophobic renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin, fibromatosis, epithelioma, epithelial disorders, Ewing tumors, extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, hard bone, gallbladder, and fibroplastic dysplasia of cholangiocarcinoma, gastric cancer, gastrointestinal, gestational trophoblastic disease, germ cell tumors, glandular dysfunction, head and neck cancer, hypothalamus, intestinal cancer, islet cell tumors, Kaposi sarcoma, kidney cancer (nephroblastoma, papillary renal cell carcinoma), leukemia, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, liver cancer (hepatoblastoma, hepatocellular carcinoma), lymphoma, lung cancer (small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc.), macrophagal disorders, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndromes, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumors, pediatric cancers, peripheral nerve sheath tumors, pheochromocytoma, pituitary tumors, prostate cancer, posterior uveal melanoma, rare blood disorders, renal metastatic cancer, rhabdoid tumors, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, interstitial disorders, synovial sarcoma, testicular cancer, thymic cancer, thymoma, thyroid metastatic cancer, and uterine cancer (cervical cancer, endometrial cancer, and leiomyoma).

[0164] In some embodiments, the DLL3-binding protein, or DLL3-targeted trispecific protein, of the present disclosure is used as a front-line treatment and administered to a subject who has not previously been treated for a cancerous condition. In other embodiments, the DLL3-targeted trispecific protein of the present disclosure is used to treat a subject who has not previously been treated, has relapsed, or has been determined to be ineffective with a previous treatment (with the DLL3-targeted trispecific protein of the present disclosure or other anti-cancer agents). In some embodiments, the DLL3-targeted trispecific protein of the present disclosure is used to treat a subject with a recurrent tumor.

[0165] In some aspects, the DLL3-binding protein, or DLL3-targeted trispecific protein, of the present disclosure is administered to treat a proliferative disease including a solid tumor, and the solid tumors include, but are not limited to, adrenal gland, liver, kidney, bladder, breast, stomach, ovary, neck, uterus, esophagus, large intestine, prostate, pancreas, lung (both small cell and non-small cell), thyroid, cytoma, sarcoma, glioblastoma, and various head and neck tumors.

[0166] In some embodiments, the DLL3 binding proteins or DLL3-targeting trispecific proteins of the present disclosure are administered to a subject suffering from melanoma. In some embodiments, the DLL3-targeting trispecific proteins of the present disclosure are used to diagnose, monitor, treat, or prevent melanoma. The term "melanoma," as used herein, includes all types of melanoma, including, but not limited to, primary melanoma, malignant melanoma, cutaneous melanoma, extracutaneous melanoma, superficial spreading melanoma, polypoid melanoma, melanoma, acanthosis nigricans, melanosarcoma, intraepidermal melanoma, nodular malignant melanoma, lentigo maligna melanoma, lentiginous melanoma, lentiginous malignant melanoma, mucosal lentiginous melanoma, mucosal melanoma, acral lentiginous melanoma, soft tissue melanoma, intraocular melanoma, invasive melanoma, familial atypical nevus melanoma (FAM-M) syndrome, desmoplastic malignant melanoma, or uveal melanoma.

[0167] DLL3 is a useful tumor marker that is expressed on many different cancers and has been shown to associate with cancer stem cells. Thus, in some embodiments in which the disclosed DLL3-binding proteins or DLL3-targeting trispecific proteins are incorporated into chimeric antigen receptors expressed on lymphocytes, the resulting "DLL3-sensitized lymphocytes" (e.g., natural killer cells or T cells that immunospecifically recognize the DLL3 determinant) can effectively mount an immune response directed against aberrant DLL3-positive cells, including cancer stem cells. This ability to effectively eliminate tumorigenic "seed" cells is often important 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 maintenance regimen following standard of care.

[0168] 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 (e.g., a T cell signaling or T cell activation domain) linked to a signaling domain. In some embodiments, a CAR comprising a DLL3-targeted trispecific binding protein of the present disclosure has the ability to redirect the specificity and reactivity of sensitized lymphocytes (e.g., T cells) towards DLL3-positive target cells in a non-MHC-restricted manner by utilizing the antigen-binding properties of an antibody or an antigen-binding fragment thereof. Non-MHC-restricted antigen recognition confers on T cells expressing the DLL3 CAR the ability to recognize tumorigenic DLL3 independent of antigen processing, and thus circumvents a major mechanism of tumor escape. Further, when expressed in T cells, it is advantageous for the CAR not to dimerize with the endogenous T cell receptor (TCR) α and β chains.

[0169] In selected embodiments, a DLL3-binding protein, or a DLL3-targeted 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 for the treatment of lung cancers including the following subtypes: small cell lung cancer, non-small cell lung cancer (e.g., squamous non-small cell lung cancer or squamous small cell lung cancer), and large cell neuroendocrine carcinoma (LCNEC).

[0170] In some embodiments, the DLL3-binding protein, or DLL3-sensitive lymphocyte, is administered to a patient presenting with a localized or advanced disease. In other embodiments, the disclosed DLL3-targeted trispecific antibody is administered to a refractory patient (i.e., a patient in whom the disease recurs during or immediately following a series of initial treatments); a sensitive patient (i.e., a patient in whom the recurrence occurs more than two to three months after the primary treatment); or a patient resistant to platinum-based agents (e.g., carboplatin, cisplatin, oxaliplatin), and / or taxanes (e.g., docetaxel, paclitaxel, larotaxel, or cabazitaxel). In another embodiment, treatment with the disclosed DLL3 CAR is effective for the treatment of ovarian cancer, including ovarian-serous carcinoma and ovarian-serous papillary carcinoma.

[0171] The DLL3-binding protein, or DLL3-targeted trispecific binding protein, of the present disclosure is used, in some embodiments, to prevent, treat, or diagnose tumors having a neuroendocrine feature or phenotype, including neuroendocrine tumors. True or reference neuroendocrine tumors (NETs) arising from the dispersed endocrine system are relatively rare, with an incidence of 2 to 5 per 100,000 people, but are highly malignant. Neuroendocrine tumors occur in the kidney, urogenital tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lung (small cell lung cancer and large cell neuroendocrine carcinoma). These tumors may secrete several hormones, including serotonin and / or chromogranin A, which can cause debilitating symptoms known as carcinoid syndrome. Such tumors are indicated by positive immunohistochemical markers such as neuron-specific enolase (NSE, also known as γ-enolase (gene symbol = ENO2)), CD56 (or NCAM1), chromogranin A (CHGA), and synaptophysin (SYP), or by genes known to show increased expression such as ASCL1. Conventional chemotherapy is not particularly effective in treating neuroendocrine tumors, and liver metastasis is a common outcome. In some embodiments, the DLL3-targeted trispecific antibody of the present disclosure is advantageously used in the treatment of neuroendocrine tumors. In some embodiments, the DLL3-targeted trispecific antibody of the present disclosure is used to treat, prevent, or diagnose pseudo-neuroendocrine tumors (pNETs) that genetically or phenotypically mimic, resemble, or exhibit traits common to canonical neuroendocrine tumors. Pseudo-neuroendocrine tumors or tumors having a neuroendocrine feature are tumors that arise abnormally from scattered neuroendocrine cells or from cells in which the neuroendocrine differentiation cascade has been reactivated during the carcinogenesis process. Such pNETs generally share certain phenotypes or biological properties with conventionally defined neuroendocrine tumors, such as the ability to produce a subset of biologically active amines, neurotransmitters, and peptide hormones.Histologically, such tumors (NETs and pNETs) have a common appearance, which includes tightly packed small cells with minimal cytoplasm of bland cytopathology and round to oval punctate nuclei. In some embodiments of the present disclosure, generally expressed histological or genetic markers used to define neuroendocrine and pseudo-neuroendocrine tumors include, but are not limited to, chromogranin A, CD56, synaptophysin, PGP9.5, ASCL1, and neuron-specific enolase (NSE). Thus, in some embodiments, the DLL3-targeted trispecific proteins, DLL3 CARs, or DLL3-sensitized lymphocytes of the present disclosure, or any combination thereof, are beneficial for treating both pseudo-neuroendocrine tumors and canonical neuroendocrine tumors, such as neuroendocrine tumors (both NETs and pNETs) that occur in the kidney, urogenital tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lung (small cell lung cancer and large cell neuroendocrine carcinoma). Further, in some embodiments, the DLL3-targeted trispecific proteins, DLL3 CARs, or DLL3-sensitized lymphocytes of the present disclosure, or any combination thereof, are used to treat tumors that express one or more markers such as NSE, CD56, synaptophysin, chromogranin A, ASCL1, or PGP9.5 (UCHL1). In some embodiments, the DLL3-targeted trispecific proteins, DLL3 CARs, or DLL3-sensitized lymphocytes of the present disclosure, or any combination thereof, are used to treat a subject bearing a tumor that is NSE+ or CD56+ or PGP9.5+ or ASCL1+ or SYP+ or CHGA+ or any combination thereof.

[0172] In another embodiment, the DLL3-targeted trispecific protein, DLL3 CAR, or DLL3-sensitized lymphocyte of the present disclosure, or any combination thereof, is used in maintenance therapy to reduce or eliminate the likelihood of tumor recurrence following the initial manifestation of the disease. In some cases, the disease has been treated and the initial tumor burden has been removed, reduced, or otherwise improved, such that the patient is asymptomatic or in remission. At such times, the subject is administered a pharmaceutically effective amount of the DLL3-binding protein of the present disclosure, the DLL3-targeted trispecific protein of the present disclosure, DLL3 CAR, or DLL3-sensitized lymphocyte, or any combination thereof, using standard diagnostic methods, regardless of whether there are few or no signs of the disease. In some embodiments, the DLL3-targeted trispecific protein, DLL3 CAR, or DLL3-sensitized lymphocyte of the present disclosure, or any combination thereof, is administered, for example, at regular intervals over a period of time, such as weekly, every two weeks, monthly, every six weeks, every two months, every three months, every six months, or annually, to reduce the likelihood of disease recurrence. Further, in some embodiments, such treatment is continued for weeks, months, years, or even an indefinite period, depending on the patient's response and clinical and diagnostic parameters.

[0173] In yet another embodiment, the DLL3 binding protein, DLL3-targeted trispecific protein, DLL3 CAR, or DLL3-sensitized lymphocyte of the present disclosure, or any combination thereof, is used prophylactically or as adjuvant therapy to prevent or reduce the likelihood of tumor metastasis after bariatric surgery. As used in the present disclosure, "bariatric surgery" is broadly defined and means any surgery, technique, or method that eliminates, reduces, treats, or improves a tumor or tumor growth. Exemplary bariatric surgeries include, but are not limited to, surgery, radiation treatment (i.e., beam radiation), chemotherapy, immunotherapy, or ablation. In some embodiments, the DLL3 binding protein, the DLL3-targeted trispecific protein of the present disclosure, the DLL3 CAR, or the DLL3-sensitized lymphocyte, or any combination thereof, is administered at an appropriate time as suggested by a clinical, diagnostic, or theranostic procedure for reducing tumor metastasis. In some embodiments, the dosing regimen involves appropriate diagnostic or monitoring techniques that allow for modification of the dosing regimen.

[0174] Still 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 at risk of developing a symptomatic but proliferative disorder. That is, in some embodiments, the DLL3 binding protein, the DLL3-targeted trispecific protein of the present disclosure, the DLL3 CAR, or the DLL3-sensitized lymphocyte, or any combination thereof, is used prophylactically and administered to patients who have been examined or tested and have one or more noted risk factors (e.g., genomic signatures, family history, in vivo or in vitro test results, etc.) but have not developed a neoplasm. In such cases, one of ordinary skill in the art could determine an effective dosing regimen by empirical observation or by accepted clinical practice.

[0175] As used herein, in some embodiments, "treatment" or "treating" or "being treated" refers to therapeutic treatment that is directed at, or delays, an undesired physiological disorder, condition, or disease, or that obtains a beneficial or desired clinical result. For purposes of this specification, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the degree of a disease, disorder, or condition; stabilization of a disease, disorder, or condition (i.e., not worsening); delay or slowing of the onset or progression of a disease, disorder, or condition; improvement of a disease, disorder, or condition state; and remission, whether detectable or undetectable, partial or total, of a disease, disorder, or condition, or enhancement or improvement thereof. Treatment includes inducing a clinically significant response without excessive levels of side effects. Treatment further includes extending survival as compared to expected survival time if not receiving treatment. In other embodiments, "treatment" or "treating" or "being treated" refers to prophylactic treatment, the purpose of which is, for example, to delay the onset or reduce the severity of an undesired physiological disease, disorder, or condition in a person predisposed to the disease (e.g., an individual having a genetic marker for a disease such as breast cancer).

[0176] In some embodiments of the methods described herein, the DLL3-binding proteins, DLL3-targeted trispecific proteins, or compositions described herein are administered in combination with a drug for the treatment of a particular disease, disorder, or condition. Drugs include, but are not limited to, therapeutic agents including antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiation therapy agents (γ-rays, X-rays, and / or targeted delivery of radioisotopes, microwaves, UV radiation, etc.), gene therapy agents (e.g., antisense, retroviral therapy, etc.), and other immunotherapeutic agents. In some embodiments, the anti-DLL3 binding proteins, or anti-DLL3-targeted trispecific proteins described herein are administered in combination with anti-diarrheal agents, anti-emetic agents, analgesics, opioids, and / or non-steroidal anti-inflammatory agents. In some embodiments, the anti-DLL3 binding proteins, or anti-DLL3-targeted trispecific proteins described herein are administered in combination with anti-cancer agents. Non-limiting examples of anti-cancer agents that can be used in various embodiments of the present disclosure, including the pharmaceutical compositions, dosage forms, and kits of the present disclosure, include the following: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldosterone; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; biseresin; bleomycin sulfate; brequinar sodium; broxuridine; busulfan; carcinoinomycin; carbetimer; carboplatin; carmustine; carboquone; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Efloxatin Hydrochloride; Elsamitrucin; Enloplatin; Emproate; Epipropidine; Epirubicin Hydrochloride; Elbuzole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Ethanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interleukin II (including recombinant interleukin II, or rIL2); Interferon α-2a; Interferon alpha-2b; Interferon α-n1; Interferon α-n3; Interferon β-Ia; Interferon γ-Ib; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Rialtrozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melenegestro acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Methopterin; Metsuldepa; Mitindomide; Mitocarcin; Mitochromin; Mitogillin; Mitomalcin; Mitomycin; Mitospir; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Pirarubicin Hydrochloride; Plicamycin; Promestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Promycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Logretimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride;Spiro masstin; Spiro platine; Streptomycin; Streptozocin; Slofenaal; Talisomycin; Tecoalan sodium; Tegafur; Trecator hydrochloride; Temoporfin; Teniposide; Teloxiron; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thiazofurin; Tirabazamine; Toremifene citrate; Tresterone acetate; Triscivilin phosphate; Trimethoprim; Trimethoprim glucuronate; Triptorelin; Tubulozole hydrochloride; Uracil mustard; Uredepa; Bapreotide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Vinepidine sulfate; Vinglycinate sulfate; Vinleurosine sulfate; Vinorelbine tartrate; Vinzolidine sulfate; Vinzolidine sulfate; Borozole; Zeniplatin; Dinostatin; Zorubicin hydrochloride. Other examples of anticancer agents include, but are not limited to, 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; Abiraterone; Aclarubicin; Alkylfulvene; Adesipenol; Adzelesin; Aldesleukin; ALL-TK antagonist; Altretamine; Ambamastin; Amidox; Amifostine; Aminolevulinic acid; Amrubicin; Amsacrine; Anagrelide; Anastrozole; Andrographolide; Angiogenesis inhibitor; Antagonist D; Antagonist G; Antarelix; Anti-dorsalizing morphogenetic protein-1; Anti-androgen, prostate cancer; Anti-estrogen; Antineoplaston; Antisense oligonucleotide; Aphidicolin glycinat; Apoptosis gene modulator; Cell death regulator; Aprinic acid; Congo red inco CDP-DL-PTBA; Arginine deaminase; Asracrine; Atamestane; Attrimastin; Axinasatatin 1; Axinasatatin 2; Axinasatatin 3; Azasetron; Azatoxin; Azatyrosine; Paclitaxel derivative; Baranol; Batimastat; BCR / ABL antagonist; Benzothirolin; Benzoyl staurosporine; Beta-lactam derivative; Beta-aretin; Betaclamycin B; Betulinic acid; bFGF inhibitor; Bicalutamide; Bisantrene; Bisaziridinyl spermine;Bianthraquinone; Bistratene A; Biserein; Breflate; Broxuridine; Budotitane; Buthionine Sulfoximine; Calcipotriol; Calphostin C; Camptothecin Derivative; Canaripox IL-2; Capecitabine; Carboxamide-Amino-Triazole; Carboxamido Triazole; CaRest M3; CARN 700; Cartilage-Derived Inhibitor; Calzeclin; Casein Kinase Inhibitor (ICOS); Castanospermine; Cecropin B; Cetrorelix; Chlorin; Chlorquinoxaline Sulfonamide; Cicaprost; cis-Porphyrin; Cladribine; Clomifene Analogue; Clotrimazole; Colismycin A; Colismycin B; Combretastatin A4; Combretastatin Analogue; Conagenin; Crambescidin 816; Crisnatol; Cryptophycin 8; Cryptophycin A Derivative; Classin A; Cyclopentanthraquinones; Cycloplatam; Cipemamycin; Cytarabine Oxaphosphate; Cytotoxic Factor; Cytostatic; Daclizumab; Decitabine; Dehydrodidemnin B; Deslorelin; Dexamethasone; Dexifosdamide; Dexrazoxane; Dexverapamil; Diadicon; Didemnin B; Dodox; Diethylnorspermine; Dihydro-5-Azacytidine; Dihydrotaxol, 9-; Dioxamycin; Diphenylspirodrimastin; Docetaxel; Docosanol; Dolasetron; Doxifluridine; Droloxifene; Dronabinol; Zouocarmycin SA; Ebselen; Ecomustine; Edelfosine; Edrecolomab; Eflornithine; Element; Emitefur; Epirubicin; Epristeride; Estramustine Analogue; Estrogen Agonist; Estrogen Antagonist; Ethanidazole; Etoposide Phosphate; Exemestane; Fadrozole; Fazarabine; Fenretinide; Filgrastim; Finasteride; Flavopiridol; Fretterastatin; Fluasterone; Fludarabine; Fluorodaunorunicin Hydrochloride; Formestane; Fostriecin; Hostemustine; Gadolinium Texaphyrin; Gallium Nitrate; Gallocitabine; Ganirelix; Gelatinase Inhibitor; Gemcitabine; Glutathione Inhibitor; Hapsulfam;Heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilmostat; imidazoacridone; imiquimod; immunostimulatory peptide; insulin-like growth factor I receptor inhibitor; interferon agonist; interferon; interleukin; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; ilsogladine; isobenzagol; isohomohalicondrin B; itacetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; reanamycin; lenograstim; sulfuric acid lentinan; reptorastatin; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprorelin + estrogen + progesterone; leuprolide; levamisole; liarozole; linear polyamine analog; lipophilic disaccharide peptide; lipophilic platinum compound; lissoclinamide 7; lobaplatin; lombricin; lometrexol; lonidamine; losoxantrone; HMG-CoA reductase inhibitor; (but not limited to, lovastatin, pravastatin, fluvastatin, simvastatin, and atorvastatin, etc.) loxoribine; larotrectin; lutetium texaphyrin; lysophyllin; cytolytic peptide; maytansine; mannostatin A; marimastat; masoprocol; maspin; matrix metalloproteinase inhibitor; matrilysin inhibitor; menogaril; melvalone; metallirin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; millimostim; mismatched double-stranded RNA; mitoguazone; mitolactol; mitomycin analog; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody (human placental gonadotropin); monophosphoryl lipid A + mycobacterial cell wall sk; mopidamol; multidrug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mikapexide B; mycobacterium cell wall extract; milapolone; n-acetyl dinarine; N-substituted benzamide; naphthalene; nagrestip; naloxone + pentazocine; napabucasin;Naftopidil; Nartograstim; Nedaplatin; Nemorubicin; Neeridronic acid; Neutral endopeptidase; Nilutamide; Nisamycin; Nitric oxide modulator; Nitroxide antioxidant; Nitrilin; O6-Benzylguanamine; Octreotide; Oxenone; Oligonucleotide; Onapristone; O; ondansetron; ondansetron; oracin; oral cytokine inducer; olmaplatin; osaterone; oxaliplatin; ogusamycin; paclitaxel; paclitaxel analog; paclitaxel derivative; parauramine; palmitoyl lysophosphatidylcholine; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; perdiccin; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenyl acetate; phosphatase inhibitor; picibanil; pilocarpine hydrochloride; pirarubicin; pirimethamine; withaferin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compound; platinum triamine complex; porfimer sodium; porphyrinomycin; prednisone; propylbis-acridone; prostaglandin J2; proteasome inhibitor; protein A-based immunomodulator; protein kinase C inhibitor; protein kinase C inhibitor, microalgae; tyrosine phosphatase protein inhibitor; purine nucleoside phosphorylase inhibitor; purpurin; pyrazoloacridine; pyridoxylated hemoglobin-polyoxyethylene conjugate; raf antagonist; raltitrexed; ramsetron; ras farnesyl protein transferase inhibitor; ras inhibitor; ras-GAP inhibitor; demethylated leptitide; rhenium Re 186 etidronate; lysophosphatidylcholine; ribozyme; RII retinamide; logretimide; rohitukine; romurtide; roquinimex; ravidinone B1; rabocil; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetic; semustine; senescence-derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction modulator; single-chain antigen-binding protein; schizophyllan; sobuzoxane; sodium borocaptate; sodium phenylacetate; salvador; somatomedin-binding protein; sonermin; sparfosic acid; spicamycin D; spiroasterol; squalenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitor; stipiamide;Stromelysin inhibitor; Sulfinosine; Superactive vasoactive intestinal peptide antagonist; Saladistin; Slamin; Swainsonine; Synthetic glycosaminoglycan; Talimustine; Tamoxifen methiodide; Tauromustine; Tazarotene; Tegafur sodium; Tegafur; Telrapirium; Telomerase inhibitor; Temoporfin; Temozolomide; Teniposide; Tetrachlorodecaoxide; Tetrazomine; Salibrastine; Thiocholin; Thrombopoietin; Thrombopoietin mimetic; Timalphazine; Thymopoietin receptor agonist; Timotrinan; Thyroid stimulating hormone; Tin ethyl etioproprin; Tiraparazamine; Titanium chloride; Topseptin; Toremifene; Pluripotent stem cell factor; Translation inhibitor; Tretinoin; Triacetyluridine; Trisciribine; Trimetrexate; Triptorelin; Tropisetron; Tulosteride; Tyrosine kinase inhibitor; Tilostatin; UBC inhibitor; Ubenimex; Urogenital sinus-derived growth inhibitor; Urokinase receptor antagonist; Bapreotide; Variolin B; Vector system, erythrocyte gene therapy; Veralezol; Veramine; American cockroach; Verteporfin; Vinorelbine; Vinzaltine; Vitaxin (registered trademark); Boroazol; Zanoterone; Zeniplatin; Zilascorb; and, Dinostatin stimalamer. Additional anti-cancer agents are 5-fluorouracil and leucovorin. These two drugs are particularly useful when used in a manner that uses thalidomide and topoisomerase inhibitors. In some embodiments, the DLL3-targeted trispecific protein of the present disclosure is used in combination with gemcitabine. In some embodiments, the DLL3-targeted trispecific protein described herein is administered before, during, or after surgery.;

[0177] Method for detecting DLL3 expression and diagnosing DLL3-related cancer According to another embodiment of the present disclosure, a kit for detecting DLL3 expression in vitro or in vivo is provided. The kit includes the aforementioned DLL3-binding protein, a DLL3-targeting trispecific protein (e.g., a trispecific protein including a labeled anti-DLL3 single-domain antibody or an antigen-binding fragment thereof), and one or more compounds for detecting the label. In some embodiments, the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

[0178] In some cases, DLL3 expression is detected in a biological sample. The sample may be any sample including, but not limited to, tissue from a biopsy, autopsy, and pathological specimen. The biological sample also includes tissue sections, e.g., frozen sections obtained for histological purposes. The biological sample further includes body fluids such as blood, serum, plasma, saliva, cerebrospinal fluid, or urine. The biological sample is typically obtained from a mammal such as a human or non-human primate.

[0179] In one embodiment, a method is provided for determining whether a subject has cancer by contacting a sample of the subject with an anti-DLL3 single-domain antibody or an anti-DLL3 trispecific protein disclosed herein; and detecting the binding of the single-domain antibody to the sample. An increase in the binding of the antibody to the sample as compared to the binding of the antibody to a control sample identifies the subject as having cancer.

[0180] In another embodiment, a method is provided for confirming a diagnosis of cancer in a subject by contacting a sample of the subject diagnosed with cancer with an anti-DLL3 single-domain antibody or an anti-DLL3 trispecific protein disclosed herein; and detecting the binding of the antibody to the sample. An increase in the binding of the antibody to the sample as compared to the binding of the antibody to a control sample confirms the diagnosis of cancer in the subject.

[0181] In some examples of the methods of the present disclosure, the DLL3-binding protein, or the DLL3-binding single domain antibody of the trispecific protein, is directly labeled. In some examples, the method further comprises contacting a second antibody that specifically binds to the anti-DLL3 single domain antibody or anti-DLL3 trispecific protein with the sample; and detecting the binding of the second antibody. An increase in the binding of the second antibody to the sample as compared to the binding of the second antibody to the control sample indicates that cancer is detected in the subject or that the diagnosis of cancer in the subject is confirmed. In some cases, the cancer is neuroendocrine cancer, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple negative breast cancer, or ovarian cancer, or other types of cancer that express DLL3. In some examples, the control sample is a sample from a subject without cancer. In certain examples, the sample is a blood sample or a tissue sample.

[0182] In some cases, the antibody that binds to (e.g., specifically binds to) DLL3 is directly labeled with a detectable label. In another embodiment, the antibody that binds to (e.g., specifically binds to) DLL3 (the first antibody) is not labeled, and another molecule that can bind to a second antibody or an antibody that specifically binds to DLL3 is labeled. The second antibody is selected to be able to specifically bind to a particular species and class of the first antibody. For example, if the first antibody is llama IgG, the secondary antibody can be anti - llama IgG. Other molecules that can bind to an antibody include, without limitation, protein A and G proteins, both of which are commercially available. Suitable labels for the antibody or secondary antibody are those described above and include various enzymes, avidin - biotin families, fluorescent materials, luminescent materials, magnetic agents, and radioactive substances. Non - limiting examples of suitable enzymes include alkaline phosphatase, β - galactosidase, or acetylcholinesterase, including horseradish peroxidase. Non - limiting examples of suitable avidin - biotin family complexes include streptavidin / biotin and avidin / biotin. Non - limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, fluorescein, dansyl chloride, or phycoerythrin. A non - limiting exemplary luminescent material is luminol, a non - limiting exemplary magnetic agent is gadolinium, and non - limiting exemplary radioactive labels include 125I, 131I, 35S, or 3H.

[0183] In an alternative embodiment, DLL3 can be analyzed in a biological sample by a competitive immunoassay that utilizes a DLL3 reference substance labeled with a detectable substance and an unlabeled antibody that specifically binds to DLL3. In this assay, the biological sample, the labeled DLL3 reference substance, and the antibody that specifically binds to DLL3 are combined, and the amount of labeled DLL3 standard that binds to the unlabeled antibody is determined. The amount of DLL3 in the biological sample is inversely proportional to the amount of labeled DLL3 standard that binds to the antibody that specifically binds to DLL3.

[0184] The immunoassays and methods disclosed herein can be used for many purposes. In one embodiment, an antibody that specifically binds to DLL3 may be used to detect the production of DLL3 in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of DLL3 in a biological sample such as a tissue sample or in a blood sample or serum sample. In some examples, DLL3 is cell surface DLL3. In other examples, DLL3 is soluble DLL3 (e.g., DLL3 or soluble DLL3 in the cell culture supernatant or in a body fluid sample such as a blood sample or serum sample).

[0185] In one embodiment, a kit is provided for detecting DLL3 in a biological sample such as a blood sample or a tissue sample. For example, to confirm a cancer diagnosis in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample can be obtained and the presence of soluble DLL3 protein or fragment can be detected. A kit for detecting a polypeptide typically includes a single domain antibody according to the present disclosure that specifically binds to DLL3. In some embodiments, an antibody fragment such as an scFv fragment, a VH domain, or a Fab is included in the kit. In further embodiments, the antibody is labeled (e.g., using a fluorescent label, a radioactive label, or an enzyme label).

[0186] In one embodiment, the kit includes a teaching material that discloses a method of using an antibody that binds to DLL3. For example, the teaching material may be written in an electronic form (such as a computer disk or a compact disk), or may be visual (such as a video file), or may be provided via an electronic network, such as the Internet, the World Wide Web, an intranet, or other networks. The kit may further include additional components to facilitate the particular use for which the kit is designed. Thus, for example, the kit may further include means for detecting a label (such as an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, or a suitable secondary label such as a secondary antibody). The kit may further include a buffer and other reagents conventionally used to carry out a particular method. Such kits and appropriate contents are well known to those skilled in the art.

[0187] In one embodiment, the diagnostic kit includes an immunoassay. The details of the immunoassay may vary depending on the particular format used, but a method for detecting DLL3 in a biological sample generally includes contacting the biological sample with an antibody that specifically reacts with the DLL3 polypeptide under immunologically reactive conditions. The antibody specifically binds under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (the bound antibody) is detected either directly or indirectly.

[0188] Methods for determining the presence or absence of cell surface markers are known in the art. For example, antibodies can bind to other compounds including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorescent dyes, metal compounds, radioactive compounds, or drugs. Antibodies can also be used in immunoassays such as, but not limited to, radioimmunoassay (RIA), ELISA, or immunohistochemical assays. Antibodies can also be used for fluorescence-activated cell sorting (FACS). FACS uses multiple color channels, low-angle and obtuse-angle light scatter detection channels, and impedance channels for a higher level of detection for separating or sorting cells (see U.S. Patent No. 5,061,620). Any of the single-domain antibodies that bind to DLL3 disclosed herein can be used in these assays. Thus, antibodies can be used in conventional immunoassays including, without limitation, ELISA, RIA, FACS, tissue immunohistochemical examination, Western blot, or immunoprecipitation.

Example

[0189] Example 1: Screening of a phage display library for identification of a DLL3-binding domain Rams were immunized with purified DLL3 protein expressed in EXPI293 (trademark) cells. A phage display library for the expression of heavy chain variable antibody domains was 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 Escherichia coli, preparing periplasmic extracts, and screening the clones for DLL3 binding activity by ELISA. In the ELISA screening (SEQ ID NO.1 - 52), 52 unique single - domain antibodies of only the heavy chain that generated a signal were identified. The sequences of CDR1, CDR2, and CDR3 of these heavy - chain variable domains were SEQ ID NO.443 - 494, SEQ ID NO.885 - 936, and SEQ ID NO.1327 - 1378, respectively.

[0190] Example 2: Humanization of DLL3 - binding single - domain antibodies and T - cell - dependent cytotoxicity assay Exemplary llama anti - DLL3 single - domain antibodies of heavy chain only from Examples 1 to 34 (SEQ ID NO.53 - 86) were humanized. The sequences of CDR1, CDR2, and CDR3 of the 34 heavy - chain single - domain antibodies were SEQ ID NO.495 - 528, SEQ ID NO.937 - 970, and SEQ ID NO.1379 - 1412, respectively.

[0191] In an expression construct comprising a signal domain, followed by the variable domain of the anti-DLL3 heavy chain only, followed by the GGGGSGGGS linker (SEQ ID NO.1808), followed by the anti-human albumin single domain antibody 10G (SEQ ID NO.1774), followed by the GGGGSGGGS linker (SEQ ID NO.1808), followed by the anti-human CD3 antibody 2B2 (SEQ ID NO.1793), followed by the HHHHHH tag (SEQ ID NO.1819), the humanized anti-DLL3 sequence was cloned into an expression vector to generate an anti-DLL3 trispecific construct.

[0192] Next, the anti-DLL3 trispecific construct containing the humanized anti-DLL3 binding sequence was transfected into EXPI293™ cells. These anti-DLL3 trispecific constructs were manipulated using the Protein A binding site, and the amount of the anti-DLL3 trispecific construct in the conditioned medium from the transfected EXPI293™ cells was quantified using an Octet instrument equipped with a Protein A chip. A trispecific protein of the same molecular weight as the anti-DLL3 trispecific protein was used as a reference.

[0193] Using conditioned media containing known concentrations of the anti-DLL3 trispecific protein, the binding affinity of the anti-DLL3 trispecific protein for human and cynomolgus monkey DLL3 proteins was measured, and the measurement was performed using an Octet instrument equipped with an anti-human Fc chip using a method of expressing the DLL3 protein as a human IgG1-Fc fusion. A single 50 nM concentration of the anti-DLL3 trispecific protein was used to perform the measurement, which enabled rank ordering based on potency. The relative affinities measured as described above are shown in Table 1. All sequences were found to bind to human DLL3 with relative affinities (K D measurements were performed, which enabled rank ordering based on potency. The relative affinities measured as described above are shown in Table 1. All sequences were found to bind to human DLL3 with relative affinities (K D ) in the range of 0.5 - 42 nM. Some of the sequences were found to bind to cynomolgus monkey DLL3 with an affinity similar to that for human DLL3, and the relative affinities of their binding to cynomolgus monkey DLL3 are shown in Table 1.

[0194] The conditioned medium was also tested in a T cell-dependent cytotoxicity assay (see Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27). In this assay, luciferase-labeled DMS-153 cells (small cell lung cancer cell line; ATCC No. ATCC® CRL-2064™) were combined with purified human T cells from a donor, and titrations of the anti-DLL3 trispecific protein were tested.

[0195] The hypothesis was made that the viability of DMS-153 cells, as determined by performing a luciferase assay 48 hours after the start of the experiment, should decrease if the anti-DLL3 trispecific protein leads T cells to kill DMS-153 cells that express DLL3.

[0196] As shown in FIG. 2-6 showing graphs of representative TDCC data, several exemplary anti-DLL3 trispecific proteins were able to reduce the viability of DMS-153 cells. FIG. 2 shows the results of TDCC assays for anti-DLL3 trispecific proteins containing 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-DLL3 trispecific proteins containing 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 containing 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 containing 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 containing the DLL3 binding domains DH58 (SEQ ID NO. 74) and DH70 (SEQ ID NO. 79). The negative control for the TDCC assay was a trispecific protein that targeted GFP instead of DLL3 (as shown in FIG. 6) in which T cells were not directed to kill DMS-153 cells. The EC 50 values are also listed in Table 1. These values were in the range of 69 pM to 11 nM.

[0197]

Table 1

[0198] Example 3: Screening of a phage display library for the identification of DLL3-binding domains with higher binding affinity using two humanized DLL3 single domain antibodies from the foregoing examples Two of the humanized antibody sequences, DH43 (SEQ ID NO.68) and DH6 (SEQ ID NO.75), were used as a starting point for creating a phage display library (according to the method described in WO2016187101A2). Subsequently, to generate an anti-DLL3 trispecific protein, the anti-DLL3 sequence from this panning was cloned into an expression vector in an expression construct containing a signal domain, followed by the variable domain of only the anti-DLL3 heavy chain, followed by the GGGGSGGGS linker (SEQ ID NO.1808), followed by an anti-human albumin single domain antibody domain, followed by the GGGGSGGGS linker (SEQ ID NO.1808), followed by an anti-human CD3 antibody fragment, and followed by the HHHHHH tag (SEQ ID NO.1819). These constructs were transfected into EXPI293™ cells, and the expressed anti-DLL3 trispecific protein was quantified as described in Example 2. The sequences of the clones identified from the panning are SEQ ID NOs. 87 - 367. Table 2 provides the CDR mutations obtained in the DH43 DLL3 binder sequences after phage display selection. Three of the clones identified from the panning, SEQ ID NOs. 199 (2E05), 330 (4D09), and 365 (4H011), were engineered to generate mutants, where each mutant had a single amino acid change from the parental sequence, for example, to remove the potential metabolic propensity of the parental sequence. In particular, the DLL3 binding domains containing SEQ ID NOs. 227 (2E05-M106Y), 228 (2E05-M106Q) were engineered mutants of SEQ ID NO.199 (2E05); SEQ ID NO.366 (4D09-M34L) was an engineered mutant of SEQ ID NO.330 (4D09); and SEQ ID NO.367 (4H11-M34L) was an engineered mutant of SEQ ID NO.365 (4H011).The CDR1 sequences of these DLL3-binding clones identified by panning are SEQ ID NOs. 529 to 809, the CDR2 sequences of the DLL3-binding clones identified by panning are SEQ ID NOs. 971 to 1251, and the CDR3 sequences of the DLL3-binding clones identified by panning are SEQ ID NOs. 1413 to 1691.

[0199] [Table 2]

[0200] Conditioned media containing known concentrations of anti-DLL3 trispecific protein was used to measure the binding affinity of the anti-DLL3 trispecific protein to human DLL3 protein using a biotinylated version of the human DLL3 protein expressed as a human IgG1 fusion protein. Binding affinity measurements were performed on an Octet instrument equipped with a streptavidin chip. A single 50 nM concentration of anti-DLL3 trispecific protein was used to measure the binding affinity of the anti-DLL3 trispecific protein to human DLL3 protein. D Measurements were performed, which allowed for a ranking of potencies. In this experiment, the relative K D The values ranged from 2.3 nM to 64 nM, as shown in Table 3. The parental binders DH43 and DH6 had K values of 7.7 ± 0.6 nM and 9.9 ± 0.3 nM, respectively, based on four samples of conditioned medium from four transfections. D had value.

[0201] To select the DLL3 binder molecules identified in this round of panning, as well as the parental DLL3 binders DH43 and DH6, more accurate affinity measurements of human DLL3 were performed using anti-DLL3 trispecific proteins at concentrations of 60 nM, 20 nM, 6.67 nM, and 2.22 nM. In addition, relative affinity measurements were performed using only 60 nM anti-DLL3 trispecific protein. The binding affinities determined from more accurate measurements of specific anti-DLL3 binding molecules are listed in Table 4 [1H012 (SEQ ID No.162); 1A011(SEQ ID No.95); 2E05(SEQ ID No.199); 4H011(SEQ ID No.365); 3C04(SEQ ID No.251); 2E02(SEQ ID No.198); 2H02(SEQ ID No.221); 3A011(SEQ ID No.238); 3A02(SEQ ID No.230); 4D09(SEQ ID No.330); DH43(SEQ ID No.68); and, DH6(SEQ ID No.75)]. In this test, the parental binder (DH43) had a K D value of 8.9 nM, while the daughter molecule with the highest affinity, 1H012 (SEQ ID NO.162), had an affinity of 2.9 nM. Furthermore, 1H012 (SEQ ID NO.162) retained the ability to bind to cynomolgus DLL3. Additionally, in this test, the parental binder (DH6) had a K D value of 9.0 nM, while the daughter molecule with the highest affinity, 4H011 (SEQ ID NO.365), had an affinity of 3.9 nM. Furthermore, 4H011 (SEQ ID NO.365) retained the ability to bind to cynomolgus DLL3.

[0202] Twenty-two DLL3 binder molecules identified in this round of panning were selected and tested in a TDCC assay using DMS-153 cells using the same protocol as described in Example 2. Exemplary TDCC data are plotted as graphs in FIGS. 7-11, EC 50The summary of the values is shown in Table 5. In this experiment, the parental DLL3 molecules (DH43 and DH6) had EC 50 values of 200 nM and 340 nM, respectively. The most potent daughter molecule of DH43 was 1H012 (SEQ ID NO. 162), with an EC 50 value of 28 nM, showing an increase more than 7-fold in TDCC potency compared to DH43, the parental DLL3 binder. The most potent daughter molecule of DH6 was 4H011 (SEQ ID NO. 365), with an EC 50 value of 36 nM, thereby showing an increase more than 8-fold in TDCC potency compared to the parental DLL3 binder molecule. The GFP-targeting control trispecific protein used as a control had no activity in this assay (as shown in Figure 11).

[0203]

Table 3-1

[0204]

Table 3-2

[0205]

Table 4

[0206]

Table 5

[0207] Example 4: Cloning of DLL3-binding molecules selected from Example 3 into mammalian cells The anti-DLL3 trispecific proteins described in Example 3, as well as the parental DLL3 binder molecules, were subcloned into a CHO cell expression vector and stably transfected in CHO cells (see Running Deer and Allison 2004. Biotechnol. Prog. 20:880 - 889). The DLL3 binder molecules were as follows: 2E05 - M106Q (SEQ ID No. 228); 2C04 (SEQ ID No. 181); 4D09 - M34L (SEQ ID No. 366); 4D09 (SEQ ID No. 330); 2E05 - M106Y (SEQ ID No. 227); 1H012 (SEQ ID No. 162) (also referred to herein as 1H12); 2E05 (SEQ ID No. 199); 2H02 (SEQ ID No. 221); 4D011 (SEQ ID No. 332) (also referred to herein as 4D11); 2E02 (SEQ ID No. 198); 4H11 - M34L (SEQ ID No. 367); 1A011 (SEQ ID No. 95) (also referred to herein as 1A11); DH6 (SEQ ID No. 75); and DH43 (SEQ ID No. 68). The anti-DLL3 trispecific proteins were purified from the conditioned medium of CHO cells in a pool of stable clones after expression using protein A and ion exchange chromatography. The purified proteins were tested in the TDCC assay using the same method as described in Example 2. The EC 50 values from the TDCC assay of this example are shown in Table 6, and a graph of the data is shown in Figures 12 - 15. The most potent molecule (2E05 - M106Q (SEQ ID NO. 228)) has an EC 50 value of 41 nM and is 6.6-fold more potent than the parental molecule (DH43). The most potent molecule derived from DH6 is 4D09 - M34L (SEQ ID NO. 366), which has an EC 50 value of 54 nM and is 4.4-fold more potent than the parental molecule (DH6).

[0208]

Table 6

[0209] Example 5: Affinity Maturation to Obtain an Anti-DLL3 Binder with Improved Affinity To obtain a more potent anti-DLL3 binder, a second round of affinity maturation was performed. A phage display library was created based on the parental sequences of DH6 (SEQ ID NO.75) and DH58 (SEQ ID NO.74). The sequences of the binders from this round of affinity maturation are provided in SEQ ID NOs. 368 to 442. The CDR1 sequences of the DLL3 binders identified from this round of affinity maturation are SEQ ID NOs. 810 to 884, the CDR2 sequences of the DLL3 binders identified from this round of affinity maturation are SEQ ID NOs. 1252 to 1326, and the CDR3 sequences of the DLL3 binders identified from this round of affinity maturation are SEQ ID NOs. 1692 to 1768. Table 7 provides the CDR mutations obtained with the DH6 DLL3 binder sequences after phage display selection.

[0210] In an expression construct comprising a signal domain, followed by an anti-DLL3 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 an HHHHHH tag (SEQ ID NO.1819), the affinity matured anti-DLL3 sequence identified as above was cloned into an expression vector to generate an anti-DLL3 trispecific construct.

[0211] Next, an anti-DLL3 trispecific construct containing an affinity matured anti-DLL3 binding sequence was transfected into EXPI293™ cells. These anti-DLL3 trispecific constructs were then engineered using Protein A binding sites, and the amount of anti-DLL3 trispecific construct in the conditioned media from the transfected EXPI293™ cells was quantified using an Octet instrument equipped with Protein A tips. A control trispecific protein of similar molecular weight to the anti-DLL3 trispecific protein was used as a reference.

[0212] The relative binding affinity of the anti-DLL3 trispecific protein to human DLL3 protein was measured using a method of expressing a biotinylated version of human DLL3 protein as a human IgG1 fusion protein using conditioned media having a known concentration of anti-DLL3 trispecific protein, and the binding affinity measurement was performed on an Octet instrument equipped with a streptavidin tip. Measurements were made using a single 50 nM concentration of anti-DLL3 trispecific protein, which enabled ranking of potencies. The measured affinities are tabulated in Table 8. All of the sequences tested were found to bind to human DLL3 with KD values in the range of 0.3 nM to 34 nM. D Measurements were made, which enabled ranking of potencies. The measured affinities are tabulated in Table 8. All of the sequences tested were found to bind to human DLL3 with KD values in the range of 0.3 nM to 34 nM. D values in the range of 0.3 nM to 34 nM.

[0213] The conditioned medium was also tested in a T-cell dependent cytotoxicity assay (Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27). In this assay, luciferase-labeled DMS-153 cells were combined with purified human T cells, and titrations of the anti-DLL3 trispecific protein were performed. It was hypothesized that if the anti-DLL3 trispecific protein directs T cells to kill DMS-153 cells expressing DLL3, the viability of DMS-153 cells, determined by performing a luciferase assay 48 hours after the start of the experiment, should decrease. Figure 16 shows graphs of representative TDCC data for anti-DLL3 trispecific proteins containing the following DLL3 binding domains: 51A02 (SEQ ID No. 409), 51G02 (SEQ ID No. 425), 52B01 (SEQ ID No. 430), 52C04 (SEQ ID No. 431), 51A05 (SEQ ID No. 411), 52D04 (SEQ ID No. 432), 51E05 (SEQ ID No. 420), 51H05 (SEQ ID No. 429), as well as purified DH43 protein (SEQ ID No. 68), and purified DH6 protein (SEQ ID No. 75). EC 50 values from the TDCC assay are shown in Table 9. The above values were in the range of 4.2 pM to 1.5 nM. The negative control for the TDCC assay was a trispecific protein that did not direct T cells to kill DMS-153 cells and targeted GFP instead of DLL3 (as shown in Figure 16).

[0214]

Table 7

[0215]

Table 8-1

[0216]

Table 8-2

[0217]

Table 8-3

[0218]

Table 9-1

[0219]

Table 9-2

[0220]

Table 9-3

[0221] Example 6: Affinity Maturation to Obtain an Anti-DLL3 Binder with Improved Affinity A specific anti-DLL3 trispecific protein containing the DLL3-binding sequence that had the most potent TDCC activity in the assay described in Example 5 and an anti-DLL3 trispecific protein containing the parental DLL3 binder DH6 were subcloned into a CHO cell expression vector and stably transfected into CHO cells (see Running Deer and Allison 2004. Biotechnol. Prog. 20:880-889). The DLL3-binding sequences were as follows: DH6 (SEQ ID No. 75); 51A2 (SEQ ID No. 408); 51A5 (SEQ ID No. 411); 51F3 (SEQ ID No. 423); 51G2 (SEQ ID No. 425); 51G10 (SEQ ID No. 427); 51H5 (SEQ ID No. 429); 51X5 (SEQ ID No. 1886); 52B1 (SEQ ID No. 430); 52C4 (SEQ ID No. 431); and 52D4 (SEQ ID No. 432). The trispecific proteins were purified from the pool of stable clones into the conditioned medium using protein A and ion exchange chromatography. The SDS-PAGE image of the purified protein is provided in Figure 17.

[0222] The affinity of human and cynomolgus monkey DLL3 was measured using biotinylated DLL3-targeting trispecific proteins at concentrations of 60 nM, 20 nM, 6.67 nM, and 2.22 nM immobilized on an octet streptavidin chip. The affinities determined from the above measurements are shown in Table 10. In this experiment, the anti-DLL3 trispecific containing DH6, which is the parental DLL3 binder sequence for the affinity matured DLL3 binder sequence, had a K D value of 13.5 nM for human DLL3 and 11 nM for cynomolgus monkey DLL3. In contrast, the 10 anti-DLL3 trispecific proteins containing the affinity matured DLL3 binder molecules tested in this experiment had K D values in the range of 0.9 - 2.2 nM for human DLL3 and K DIt had a value. Therefore, the improvement in affinity was in the range of 6.1 to 15 times for human DLL3 and 3.2 to 7.9 times for cynomolgus monkey DLL3.

[0223] Purified proteins were tested in a TDCC assay using the same method as described in Example 2, except that two additional cell lines (DMS-53 and NCI-H510A) that express DLL3 were included in the assay. The EC 50 values are shown in Table 11, and graphs of the TDCC data for DMS-53 and DMS-153 are provided in Figures 18 - 19, respectively. The trispecific molecule targeting GFP had no activity in these assays (as shown in Figures 18 - 19). Compared to the parental molecule DH6, the EC 50 values were improved by 2.3 to 12.1 times in DMS-153 cells, 4.5 to 31.5 times in NCI-H510A cells, and 8.1 to 26.1 times in DMS-153 cells.

[0224]

Table 10

[0225]

Table 11

[0226] Example 7: T Cell-Dependent Cytotoxicity Assay Using an Exemplary DLL3-Targeted Trispecific Protein Comprising a DLL3-Binding Protein of the Present Disclosure Several exemplary DLL3 trispecific proteins containing the DLL3 binding domain of the present disclosure (52D04 (SEQ ID NO. 432)) were tested in a T cell-dependent cytotoxicity (TDCC) assay (see Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27), and the results are shown in FIGS. 22-24. The trispecific proteins contained a DLL3 binding domain, an albumin binding domain (anti-ALB), and a CD3 binding domain (anti-CD3) in an anti-DLL3:anti-ALB:anti-CD3 configuration (TAC) as shown in FIG. 20, or in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration as shown in FIG. 21. The TDCC assay was performed in the presence or absence of 15 mg / ml human serum albumin (HSA). In this assay, luciferase-labeled NCI-H2171 (FIG. 22), DMS-79 (FIG. 23), SHP77 (FIG. 24), or WM2664 (FIG. 25) cells were combined with purified human T cells, and titrations of exemplary DLL3-targeted trispecific proteins were performed in the presence or absence of albumin. The hypothesis was made that the viability of DMS-153 cells should decrease as determined by performing a luciferase assay 48 hours after the start of the experiment if an anti-DLL3 binding trispecific protein leads T cells to kill NCI-H2171, DMS-79, SHP77, or WM2664 cells that express DLL3. FIG. 22 shows a graph of representative TDCC data for DLL3 binding trispecific proteins in TAC or CAT configurations containing the following DLL3 binding domains using NCI-H2171 cells. FIG. 23 shows a graph of representative TDCC data for DLL3 binding trispecific proteins in TAC or CAT configurations containing the following DLL3 binding domains using DMS-79 cells. FIG. 24 shows a graph of representative TDCC data for DLL3 binding trispecific proteins in TAC or CAT configurations containing the following DLL3 binding domains using SHP77 cells.Figure 25 shows a graph of representative TDCC data of DLL3-binding trispecific proteins in the TAC or CAT configuration containing the following DLL3-binding domains, using WM2664 cells. EC of the TDCC assay. 50 values are shown in Table 12. As shown in the graph and by the EC 50 values, in the presence of human serum albumin (HSA), the DLL3-binding trispecific protein with the CAT orientation (Figure 21) was more potent in the TDCC assay than the DLL3 trispecific protein with the TAC configuration.

[0227] [Table 12]

[0228] Example 8: Binding of Exemplary DLL3-Targeted Trispecific Proteins to Human T Cells In the cell binding assay, human T cells were cultured in the presence or absence of an exemplary DLL3-targeting trispecific protein (either an anti-DLL3:anti-ALB:anti-CD3 (TAC) construct (SEQ ID NO. 1891); or an anti-CD3:anti-ALB:anti-DLL3 (CAT) construct (SEQ ID NO. 1890)). The human T cells were further cultured with a secondary antibody (anti-trispecific antibody) that could recognize the anti-albumin domain in the exemplary trispecific molecule conjugated to Alexa Fluor 647. Binding of the anti-trispecific antibody was measured by flow cytometry. Robust binding of the anti-trispecific antibody was seen in the presence of an exemplary DLL3 trispecific protein of the anti-DLL3:anti-ALB:anti-CD3 (TAC) construct (right peak in the plot in Figure 26), compared to cells cultured with the secondary antibody alone, or cells cultured without the exemplary trispecific protein or secondary antibody (left peak in the plot in Figure 26). Robust binding of the anti-trispecific antibody was seen in the presence of an exemplary DLL3 trispecific protein of the anti-CD3:anti-ALB:anti-DLL3 (CAT) construct (right peak in the plot in Figure 27), compared to cells cultured with the secondary antibody alone, or cells cultured without the exemplary trispecific protein or secondary antibody (left peak in the plot in Figure 27).

[0229] Example 9: Binding of Exemplary DLL3-Targeted Trispecific Proteins to DLL3 Expressed on Cancer Cell Lines In another binding assay, 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 cultured with an exemplary DLL3-targeting trispecific molecule (CAT or TAC construct; SEQ ID NO. 1890 and SEQ ID NO. 1891) or a control trispecific molecule targeting GFP. After incubation, the cells were washed to remove unbound trispecific molecules and further cultured with a secondary antibody capable of recognizing the anti-albumin domain in the trispecific molecule conjugated to Alexa Fluor 647 or FITC. Binding of the exemplary DLL3-targeting trispecific molecule to the cells, or binding of the control trispecific molecule to the cells, was measured by flow cytometry. Robust binding of the DLL3-targeting trispecific (TAC construct) to each cell line was observed (right peak in the plot in Figure 28) compared to cells cultured with the control trispecific molecule targeting GFP (left peak in the plot in Figure 28). Robust binding of the DLL3-targeting trispecific (CAT construct) to each cell line was also observed (right peak in the plot in Figure 29) compared to cells cultured with the control trispecific molecule targeting GFP (left peak in the plot in Figure 29). In control experiments using the cell lines HCTI16 (colon cancer cell line) and NCI-H292 (lung cancer cell line) that lack DLL3 expression, similar amounts of anti-trispecific antibody bound to cells cultured with the exemplary DLL3-targeting trispecific protein or a control trispecific molecule targeting GFP (not shown), indicating that the exemplary DLL3-targeting trispecific molecule did not bind to cells lacking DLL3 expression.

[0230] Example 10: Ability of an Exemplary DLL3-Targeting Trispecific Protein to Induce T Cell-Mediated Killing of Cancer Cell Lines Expressing DLL3 The goal of this study was to evaluate whether an exemplary DLL3-targeted trispecific molecule could direct T cells to kill cell lines expressing DLL3, NCI-H82, SHP77, DMS53, and NCI-H2171. Cells expressing DLL3 used in this study were engineered to express luciferase.

[0231] In the TDCC assay (T cell-dependent cytotoxicity assay), T cells from four healthy donors (Donor 2; Donor 47; Donor 81; Donor 86) and cells expressing DLL3 were mixed, and various amounts of an exemplary DLL3-targeted trispecific protein (CAT or TAC constructs; SEQ ID NO. 1890 and SEQ ID NO. 1891) were added to the mixture. The above mixture was cultured at 37 °C for 48 hours. As a control, a parallel experiment was performed using a control trispecific molecule targeting GFP. After 48 hours, the amount of remaining viable cells expressing DLL3 was quantified using a luminescence assay. It was observed that DLL3-targeted trispecific molecules (both TAC and CAT constructs) could efficiently direct T cells from all four healthy donors to kill all four cell lines expressing DLL3 (see Figures 30, 31, 32, and 33 for results using the TAC construct; see Figures 34, 35, 36, and 37 for results using the CAT construct), while the control GFP TriTAC molecule could not direct T cells to kill the cell lines as efficiently (also shown in Figures 30-37). EC 50 values are presented in Tables 13 and 14. Furthermore, TDCC assays were performed using DLL3-targeted TriTAC, and cell lines lacking DLL3 expression, NCI-H292, and HCT116. It was observed that DLL3-targeted TriTAC could not direct T cells to kill these two cell lines lacking DLL3 expression (not shown).

[0232]

Table 13

[0233]

Table 14

[0234] Example 11: DLL3-dependent activation of T cells by an exemplary DLL3-targeting trispecific protein In this assay, T cells and NCI-H82 cells or DMS53 cells from four different healthy donors (Donor 2; Donor 35; Donor 47; and Donor 86) were cultured with an exemplary DLL3-targeting trispecific protein (CAT or TAC constructs; SEQ ID NO. 1890 and SEQ ID NO. 1891) at 37 °C for 48 hours. T cells from the same donors were also cultured with a control trispecific molecule (GFP TriTAC) targeting GFP and NCI-H82 or DMS53 cells at 37 °C for 48 hours. After 48 hours, the T cells were harvested and the expression of CD69 and CD25 on the T cells was measured by flow cytometry. As seen in Figures 38 - 45, an increase in the expression of CD69 or CD25 was detected on the T cells of all four healthy donors in the presence of NCI-H82 cells or SHP77 cells and the DLL3-targeting trispecific molecule, but not in the presence of the negative control GFP TriTAC. Parallel experiments were performed using HCT116 cells lacking DLL3 expression. No increase in the expression of CD69 or CD25 was observed with the DLL3 trispecific molecules tested using HCT116 cells (not shown).

[0235] Example 12: DLL3-dependent cytokine production by T cells induced by an exemplary DLL3-targeting trispecific protein In this assay, T cells from healthy donors and NCI-H82 or SHP77 cells were cultured with an exemplary DLL3-targeted trispecific molecule (CAT or TAC construct; SEQ ID NO. 1890 and SEQ ID NO. 1891) at 37 °C for 48 hours. T cells from the same subject were also cultured with a control trispecific molecule (GFP TriTAC) targeting GFP and NCI-H82 or DMS53 cells at 37 °C for 48 hours. After 48 hours, the conditioned medium was harvested and the amounts of various cytokines present in the conditioned medium were measured using an electrochemiluminescence assay (Meso Scale Discovery). It was observed that IFNγ, IL-2, and TNFα were secreted into the above medium in the presence of NCI-H82 or SHP77 cells and the DLL3-targeted trispecific molecule, but not in the presence of the control GFP-targeted TriTAC molecule. For the DLL3-targeted trispecific molecule in TAC construct: the production of IFNγ is shown in FIGS. 46 and 47; the production of IL-2 is shown in FIGS. 48 and 49; the production of TNFα is shown in FIGS. 50 and 51. For the DLL3-targeted trispecific molecule in CAT construct: the production of IFNγ is shown in FIGS. 52 and 53; the production of IL-2 is shown in FIGS. 54 and 55; the production of TNFα is shown in FIGS. 56 and 57.

[0236] Example 13: Inhibition of the growth of NCI-H82 xenografts by an exemplary DLL3-targeted trispecific protein In this study, on day 0, 5×10 6 human T cells and 5×10 6NCI-H82 small cell lung cancer cells were injected into mice. On days 1 to 10, an exemplary DLL3-targeted trispecific molecule (CAT or TAC construct; SEQ ID NO. 1890 and SEQ ID NO. 1891) at a dose of 20, 100, or 500 μg / kg, or a negative control GFP-targeted TriTAC at a dose of 500 μg / kg, was injected daily into the peritoneal cavity (i.p.) of the mice. Tumor volume was measured every few days from day 7 to day 24. As shown in Figure 58, significant inhibition of tumor growth was observed in mice injected with all doses of the DLL3-targeted trispecific protein compared to mice administered the GFP-targeted TriTAC at a dose of 500 μg / kg.

[0237] Example 14: Removal of NCI-H82 xenografts by an exemplary DLL3-targeted trispecific protein In this study, on day 0, 5×10 6 NCI-H82 small cell lung cancer cells were subcutaneously injected. On day 8, the mice were randomized, and 2×10 7 human T cells per mouse were injected. On days 9 to 18, an exemplary DLL3-targeted trispecific molecule (CAT construct; SEQ ID NO. 1890) at a dose of 1, 10, or 100 μg / kg, or a negative control GFP-targeted TriTAC at a dose of 100 μg / kg, was injected daily into the peritoneal cavity of the mice. Tumor volume was measured every few days from day 8 to day 29. As shown in Figure 59, significant inhibition of tumor growth was observed in mice injected with the DLL3-targeted trispecific molecule at doses of 10 μg / kg and 100 μg / kg compared to mice administered the GFP-targeted TriTAC at a dose of 100 μg / kg.

[0238] Example 15: Inhibition of growth of SHP77 xenografts by an exemplary DLL3-targeted trispecific protein In this study, on day 0, 5×10 6 human T cells and 1×10 7SHP77 small cell lung cancer cells were injected into mice. On days 1 to 10, a DLL3-targeted trispecific molecule (CAT structure; SEQ ID NO.1890) at a dose of 1, 10, or 100 μg / kg, or a negative control GFP-targeted TriTAC at a dose of 100 μg / kg, was injected intraperitoneally into the mice daily. From day 6 to day 28, the tumor volume was measured every few days. As shown in Figure 60, significant inhibition of tumor growth was observed in the mice injected with the DLL3-targeted trispecific molecule at doses of 10 μg / kg and 100 μg / kg compared to the mice administered the GFP-targeted TriTAC at a dose of 100 μg / kg.

[0239] Example 16: Pharmacokinetic Profile of an Exemplary DLL3-Targeted Trispecific Protein The half-life of the DLL3-targeted trispecific protein when administered at 0.3 mg / kg is up to 3 days to up to 3.9 days in cynomolgus monkeys.

[0240] In this study, after an exemplary DLL3-targeted trispecific molecule (in the CAT configuration or the TAC configuration; SEQ ID No.1890 and SEQ ID No.1891) at a dose of 0.3 mg / kg was intravenously injected into cynomolgus monkeys, serum samples were collected at various time points after injection. Each dose was injected into 2 cynomolgus monkeys. The amount of the DLL3-targeted trispecific molecule in the serum was measured using an anti-idiotype antibody that recognizes the DLL3-targeted trispecific molecule in an electrochemiluminescence assay. Figure 61 shows a plot of the amount of the DLL3-targeted trispecific molecule in the serum at various time points. Subsequently, using this data, the pharmacokinetic properties of the DLL3-targeted trispecific molecule were calculated as provided in Table 15. Based on this pharmacokinetic data, a dosing schedule of once or twice a week for human subjects was contemplated.

[0241] [Table 15]

[0242] The half-life of the DLL3-targeted trispecific molecule when administered at 1 or 10 mg / kg is up to 2.8 to 3.3 days in cynomolgus monkeys.

[0243] In this study, after intravenous injection of an exemplary DLL3-targeted trispecific molecule at a dose of 1 mg / kg or 10 mg / kg into cynomolgus monkeys, serum samples were collected at various time points after injection. Each dose was injected into two cynomolgus monkeys. The amount of DLL3-targeted TriTAC in serum was measured using an anti-idiotype antibody that recognizes DLL3-targeted TriTAC in an electrochemiluminescence assay. Figure 62 shows a plot of the amount of DLL3-targeted trispecific molecule in serum at various time points. Subsequently, this data was used to calculate the pharmacokinetic properties of the TriTAC molecule as provided in Table 16. This pharmacokinetic data suggests dosing once or twice a week in humans.

[0244]

Table 16

[0245] When administered as a single dose of up to 10 mg / kg, the exemplary DLL3 trispecific protein was well tolerated in cynomolgus monkeys.

[0246] A transient increase in serum cytokine values was observed mainly when an exemplary DLL3-targeted trispecific protein (in the CAT construct) was administered at a dose of 10 mg / kg (see Figure 63. IFNγ is shown in the first panel, IL-6 in the second panel, and IL-10 in the third panel). Transient T cell marginalization and T cell activation were also observed (not shown). No macroscopic findings or differences in organ weights related to the DLL3 trispecific protein were observed at the end stage and recovery euthanasia, and no microscopic findings related to the DLL3 trispecific protein were observed at recovery euthanasia.

[0247] To demonstrate that cells harboring DLL3-targeted TriTAC led to lethal activity after administration to cynomolgus monkeys, serum samples collected from the 10 mg / kg dosing group 168 hours after administration were examined by the DMS53 TDCC assay and compared to freshly thawed DLL3-targeted TriTAC. Cytotoxicity of the same DMS53 cells was observed against both the serum samples and the freshly thawed protein (Figure 64). This indicates that DLL3-targeted TriTAC retains the ability to kill target cells in T cells up to one week after administration to cynomolgus monkeys.

[0248] Example 17: Xenograft Tumor Model Exemplary anti-DLL3-targeted trispecific proteins of the present disclosure are evaluated against a xenograft model.

[0249] Female immunodeficient NOD / scid mice are irradiated at a sublethal dose (2 Gy) and inoculated subcutaneously in the right dorsal flank with 1×10 6 NCI-H28 cells. When the tumors reach 100-200 mm 3 , the animals are assigned to three treatment groups. To groups 2 and 3 (8 animals each), 1.5×10 7 activated human T cells are injected intraperitoneally. Three days later, the animals in group 3 are subsequently administered, a total of 9 times, an exemplary DLL3 trispecific antigen-binding protein (such as 1, 10, 50, or 100 μg / kg) (qdx9d) intravenously. Groups 1 and 2 are treated with vehicle only. Body weight and tumor volume are measured for 30 days.

[0250] Tumor growth in animals treated with the exemplary DLL3-targeted trispecific proteins of the previous example is expected to be statistically significantly delayed compared to each of the vehicle-treated control groups.

[0251] Example 18: Proof-of-Concept Clinical Trial Protocol for Administration of an Exemplary DLL3 Trispecific Antigen-Binding Protein (Anti-DLL3 Trispecific Protein) to Patients with Neuroendocrine Carcinoma This study is a Phase I / II clinical trial testing an exemplary DLL3-targeted antigen-binding protein as a treatment for neuroendocrine carcinoma.

[0252] Test Outcome

[0253] Primary Outcome: Maximum Tolerated Dose of Exemplary DLL3-Targeted Trispecific Protein

[0254] Secondary Outcome: Determination of Whether the In Vitro Response of Exemplary DLL3-Targeted Trispecific Protein Is Associated with Clinical Response

[0255] Phase I

[0256] The maximum tolerated dose (MTD) will be determined in the Phase I section of this trial. 1.1 Determine the maximum tolerated dose (MTD) in the Phase I section of this trial. 1.2 To evaluate the exemplary DLL3-targeted trispecific protein, patients meeting the eligibility criteria will be enrolled in this trial. 1.3 The goal is to identify the maximum dose of the exemplary anti-DLL3 trispecific protein that can be safely administered without causing severe or intractable side effects in the participants. The dose will depend on the number of participants enrolled in previous trials and the adequacy of tolerance to said dose. Not all participants will receive the same dose.

[0257] Phase II 2.1 In the subsequent Phase II section, treatment will be performed at the MTD, and the goal will be to determine whether the response rate is at least 20% as a result of treatment with the exemplary DLL3-targeted trispecific protein. Primary Outcome for Phase II---Determination of Whether at Least 20% of Patients Achieve a Clinical Response (Rapid (blast) Response, Minor Response, Partial Response, or Complete Response) as a Result of Treatment with the Exemplary DLL3-Targeted Trispecific Protein

[0258] Eligibility: Patients proven to have neuroendocrine tumors by biopsy. This patient is positive for somatostatin receptor as demonstrated by somatostatin receptor PET.

[0259] All sites or origins are eligible.

[0260] Functional and non-functional tumors are tolerated.

[0261] Patients who are not candidates for surgical debulking.

[0262] Patients with an ECOG performance status of 0, 1, or 2.

[0263] Patients 18 years of age or older.

[0264] Patients who can understand and are willing to sign a written informed consent document.

[0265] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions are presently contemplated by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be utilized in practicing the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be embraced thereby.

[0266]

Table 17-1

[0267]

Table 17-2

[0268]

Table 17-3

[0269]

Table 17-4

[0270]

Table 17-5

[0271]

Table 17-6

[0272]

Table 17-7

[0273]

Table 17-8

[0274]

Table 17-9

[0275]

Table 17-10

[0276]

Table 17-11

[0277]

Table 17-12

[0278]

Table 17-13

[0279]

Table 17-14

[0280]

Table 17-15

[0281]

Table 18-1

[0282]

Table 18-2

[0283]

Table 18-3

[0284]

Table 18-4

[0285]

Table 18-5

[0286]

Table 18-6

[0287]

Table 18-7

[0288]

Table 18-8

[0289]

Table 18-9

[0290]

Table 18-10

[0291]

Table 19-1

[0292]

Table 19-2

[0293]

Table 19-3

[0294]

Table 19-4

[0295]

Table 19-5

[0296]

Table 19-6

[0297]

Table 19-7

[0298]

Table 19-8

[0299]

Table 19-9

[0300]

Table 19-10

[0301]

Table 20-1

[0302]

Table 20-2

[0303]

Table 20-3

[0304]

Table 20-4

[0305]

Table 20-5

[0306]

Table 20-6

[0307]

Table 20-7

[0308]

Table 20-8

[0309]

Table 20-9

[0310]

Table 20-10

[0311]

Table 21-1

[0312]

Table 21-2

[0313]

Table 22-1

[0314]

Table 22-2

[0315]

Table 22-3

[0316]

Table 23

[0317]

Table 24-1

[0318]

Table 24-2

[0319]

Table 24-3

[0320]

Table 25

Claims

Claim 1: A pharmaceutical composition comprising: (i) a DLL3-targeted trispecific protein; and (ii) a pharmaceutically acceptable carrier, wherein the DLL3-targeted trispecific protein is: (a) a first domain (A) that is a single-chain variable fragment that specifically binds to human CD3; (b) a second domain (B) that is a single domain antibody that specifically binds to human serum albumin protein; (c) a third domain (C) that is a single-domain antibody that specifically binds to a DLL3 protein, wherein the third domain (C) comprises a complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID No. 874, a CDR2 comprising the amino acid sequence of SEQ ID No. 1316, and a CDR3 comprising the amino acid sequence of SEQ ID No. 1758; A pharmaceutical composition comprising:

2. The domains of the DLL3-targeting trispecific protein are arranged in the order of H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, H2N-(C)-(B)-(A)-COOH, or H2N-(C)-(A)-(B)-COOH, or in the order of H2N-(A)-L1-(B)-L2-(C)-COOH, H2N-(A)-L1-(C)-L2-(B)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2 The pharmaceutical composition of claim 1, wherein the amino acids are linked in the order of N-(B)-L1-(C)-L2-(A)-COOH, H 2 N-(C)-L1-(B)-L2-(A)-COOH, or H 2 N-(C)-L1-(A)-L2-(B)-COOH.

3. The pharmaceutical composition described in claim 2, wherein the domains of the DLL3-targeting trispecific protein are linked in the order H2N-(A)-(B)-(C)-COOH, or in the order H2N-(A)-L1-(B)-L2-(C)-COOH by linkers L1 and L2.

4. The pharmaceutical composition described in claim 2, wherein the domains of the DLL3-targeting trispecific protein are linked in the order H2N-(C)-(B)-(A)-COOH, or in the order H2N-(C)-L1-(B)-L2-(A)-COOH by linkers L1 and L2.

5. The pharmaceutical composition of claim 2, wherein the linkers L1 and L2 are each independently selected from the group consisting of (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), or (GGGGS)n (SEQ ID No. 1814), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

6. The pharmaceutical composition of claim 2, wherein the linkers L1 and L2 independently comprise the sequence GGGGSGGGGS (SEQ ID No. 1808).

7. The pharmaceutical composition of claim 1, wherein the third domain (C) comprises an affinity-matured binding molecule derived from a parent molecule that specifically binds to the DLL3 protein.

8. The pharmaceutical composition of claim 7, wherein the affinity-matured DLL3 binding molecule has a binding affinity for the DLL3 protein that is 2 to 50 times greater than the binding affinity of the parent molecule for the DLL3 protein.

9. The pharmaceutical composition of claim 1, wherein the third domain (C) comprises the amino acid sequence of SEQ ID No.

432.

10. The pharmaceutical composition of claim 1, wherein the second domain (B) comprises a CDR1 comprising the amino acid sequence of SEQ ID No. 1783, a CDR2 comprising the amino acid sequence of SEQ ID No. 1787, and a CDR3 comprising the amino acid sequence of SEQ ID No. 1792.

11. The pharmaceutical composition of claim 1, wherein the second domain (B) comprises the amino acid sequence of SEQ ID No. 1774.

12. The pharmaceutical composition of claim 1, wherein the first domain (A) comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises an HCDR1 comprising the amino acid sequence of SEQ ID No. 1830, an HCDR2 comprising the amino acid sequence of SEQ ID No. 1840, and an HCDR3 comprising the amino acid sequence of SEQ ID No. 1842, and the VL comprises an LCDR1 comprising the amino acid sequence of SEQ ID No. 1852, an LCDR2 comprising the amino acid sequence of SEQ ID No. 1877, and an LCDR3 comprising the amino acid sequence of SEQ ID No. 1878.

13. The pharmaceutical composition of claim 1, wherein the first domain (A) comprises the amino acid sequence of SEQ ID No. 1793.

14. The pharmaceutical composition of claim 1, comprising the sequence of SEQ ID No. 1890.

15. A pharmaceutical composition for use in treating or ameliorating a DLL3-expressing cancer in a subject, said pharmaceutical composition comprising: (i) a DLL3-targeting trispecific protein; and (ii) a pharmaceutically acceptable carrier, wherein said DLL3-targeting trispecific protein is: (a) a first domain (A) that is a single-chain variable fragment that specifically binds to human CD3; (b) a second domain (B) that is a single domain antibody that specifically binds to human serum albumin protein; (c) a third domain (C) that is a single-domain antibody that specifically binds to a DLL3 protein, wherein the third domain (C) comprises a complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID No. 874, a CDR2 comprising the amino acid sequence of SEQ ID No. 1316, and a CDR3 comprising the amino acid sequence of SEQ ID No. 1758; A pharmaceutical composition comprising:

16. The pharmaceutical composition of claim 15, wherein the cancer comprises lung cancer, neuroendocrine cancer, gastric cancer, ovarian cancer, or triple-negative breast cancer.

17. The pharmaceutical composition described in claim 16, wherein (i) the lung cancer includes small cell lung cancer, or (ii) the neuroendocrine cancer includes neuroendocrine prostate cancer.

18. A pharmaceutical composition for use in treating or ameliorating a DLL3-expressing cancer in a subject, comprising: (i) a DLL3-targeting trispecific protein comprising the amino acid sequence of SEQ ID No. 1890; and (ii) a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18, wherein the cancer comprises lung cancer, neuroendocrine cancer, gastric cancer, ovarian cancer, or triple-negative breast cancer.

20. The pharmaceutical composition of claim 19, wherein (i) the lung cancer includes small cell lung cancer, or (ii) the neuroendocrine cancer includes neuroendocrine prostate cancer.

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