DLL3 binding proteins and methods of use

Abstract

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

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 736,368, filed on September 25, 2018, U.S. Provisional Application No. 62 / 736,358, filed on September 25, 2018, and U.S. Provisional Application No. 62 / 877,227, filed on July 22, 2019, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on September 25, 2019 is named 47517-733_601_SL.txt and is 951,519 bytes in size. Background Art

[0005] In a variety of clinical settings, it is often necessary to selectively destroy single cells or specific cell types. For example, the primary goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and tissues intact and undamaged. One such approach is to induce an immune response against the tumor, so that immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTL) attack and destroy tumor cells. Summary of the invention

[0006] There remains a need for other available options for treating neoplastic diseases associated with overexpression of DLL3, such as neuroendocrine tumors. In certain embodiments, the present disclosure provides single domain proteins and multispecific proteins that specifically bind to DLL3 on the surface of tumor target cells, such as trispecific proteins containing DLL3 binding domains as described herein. In some embodiments, the present disclosure provides delta-like ligand 3 (DLL3) binding proteins or multispecific proteins as described above, which can be used to diagnose and treat indications associated with the expression of DLL3.

[0007] One embodiment provides a DLL3-targeting trispecific protein, wherein the protein comprises (a) a first domain (A), which is a single-chain variable fragment that can specifically bind to human CD3; (b) a second domain (B), which is a single-domain antibody that can specifically bind to human serum albumin; and (c) a third domain (C), which is a single-domain antibody that can specifically bind to the DLL3 protein.

[0008] In some embodiments, the domains are connected 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 through 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, H2N-(C)-L1-(B)-L2-(A)-COOH or H2N-(C)-L1-(A)-L2-(B)-COOH are connected in sequence.

[0009] 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 parent molecule that specifically binds to the DLL3 protein. In some embodiments, wherein the affinity matured DLL3 binding molecule has a binding affinity for the DLL3 protein that is about 2 to about 50 times greater than the binding affinity of the parent 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 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 SEQ ID No. 1-52. In some embodiments, the third domain (C) comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises a sequence selected from SEQ ID No.443-884 and 1887, wherein the CDR2 comprises a sequence selected from SEQ ID No.885-1326 and 1888, and wherein the CDR3 comprises a sequence selected from SEQ ID No.1327-1768 and 1889. In some embodiments, the CDR1 comprises a sequence selected from SEQ ID No.495-528. In some embodiments, the CDR2 comprises a sequence selected from SEQ ID No.937-970. In some embodiments, the CDR3 comprises a sequence selected from SEQ ID No.1379-1412. In some embodiments, the third domain (C) comprises a sequence at least 80% identical to a sequence selected from SEQ ID No.53-86. In some embodiments, the CDR1 comprises a sequence selected from SEQ ID No.529-809. In some embodiments, the CDR2 comprises a sequence selected from SEQ ID No.971-1251. In some embodiments, the CDR3 comprises a sequence selected from SEQ ID No.1379-1412. In some embodiments, the third domain (C) comprises a sequence at least 80% identical to a sequence selected from SEQ ID No.87-367. In some embodiments, the CDR1 comprises a sequence selected from SEQ ID No.810-884. In some embodiments, the CDR2 comprises a sequence selected from SEQ ID No.1252-1326. In some embodiments, the CDR3 comprises a sequence selected from SEQ ID No.1692-1768. In some embodiments, the DLL3 targeting tri-specific protein according to claim 1, wherein the third domain (C) comprises a sequence at least 80% identical to a sequence selected from SEQ ID No.368-442.

[0010] One embodiment provides a DLL3-targeting trispecific protein, wherein the protein comprises (a) a first domain (A), which is a single-chain variable fragment that can specifically bind to human CD3; (b) a second domain (B), which is a single-domain antibody that can specifically bind to human serum albumin; and (c) a third domain (C), which is a single-domain antibody that can specifically bind to DLL3, wherein the third domain comprises the sequence of SEQ ID No.68 or SEQ ID No.75, or is derived from SEQID No.68 or SEQ ID No.75.

[0011] 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 the CDR1 comprises the following sequence: X1X2X3X4X5X6X7SX8A, the CDR2 comprises the following sequence: GJ1SJ2J3GJ4J5J6YJ7J8SVKG (SEQ ID No. 1894), and the CDR3 comprises the following sequence: Z1Z2Z3Z4Z5Z6Z7Z8Z9.

[0012] In some embodiments:

[0013] X1 is A, D, E, F, G, H, K, L, M, N, Q, R, S, V, W or Y;

[0014] X2 is D, E, H, K, M, P, R, S, T or Y;

[0015] X3 is A, D, G, H, K, N, P, Q, R, S, T, V or Y;

[0016] X4 is K, S or V;

[0017] X5 is A, F, G, H, K, L, M, N, Q, R, S, T, V, W or Y;

[0018] X6 is D, F, H, I, K, L, M, N, Q, R, S, V or Y;

[0019] X7 is L or M; and

[0020] X8 is I, L, M, S, T or V.

[0021] In some embodiments:

[0022] J1 is I or V;

[0023] J2 is A, D, E, G, H, I, K, L, N, P, Q, R, S, T, V or Y;

[0024] J3 is A, D, E, G, H, N, R or T;

[0025] J4 is H, P, R or S;

[0026] J5 is A, H, I, K, M, N, Q, R, T or V;

[0027] J6 is A, D, G, H, I, L, M, N, S, T, V or Y;

[0028] J7 is A, F, I, L, M, R, S, T, V or Y;

[0029] J8 is A, D, E, G, H, K, L, N, R, S or V;

[0030] In some embodiments:

[0031] Z1 is L or Y;

[0032] Z2 is D, E, G, H, K, N, Q, R, S, T, V or Y;

[0033] Z3 is Q or W;

[0034] Z4 is A, D, E, G, H, I, K, L, M, P, R, S, T or V;

[0035] Z5 is A, D, E, G, N, R, S, T or Y;

[0036] Z6 is A, P, R or S;

[0037] Z7 is A, D, F, G, H, L, M, N, Q, R, S, T, V or Y;

[0038] Z 8为 A, G, I, K, P, Q, R, S, or T; and

[0039] Z9 is F, H or Y.

[0040] In some embodiments, linkers L1 and L2 are each independently selected from (GS) n (SEQ ID No.1809), (GGS) n (SEQ ID No.1810), (GGGS) n (SEQ ID No.1811), (GGSG) n (SEQ ID No.1812), (GGSGG) n (SEQ ID No.1813) or (GGGGS) n(SEQ ID No. 1814), wherein 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 connected in the order of H2N-(A)-(B)-(C)-COOH or in the order of H2N-(A)-L1-(B)-L2-(C)-COOH through linkers L1 and L2.

[0041] One embodiment provides a DLL3 binding protein comprising the formula:

[0042] f1-r1-f2-r2-f3-r3-f4

[0043] wherein r1 is complementarity determining region 1 (CDR1) and is identical to a sequence selected from SEQ ID No.443-884 and 1887 or comprises one or more amino acid residue substitutions relative to the sequence; r2 is CDR2 and is identical to a sequence selected from SEQ ID No.885-1326 and 1888 or comprises one or more amino acid residue substitutions relative to the sequence; and r3 is CDR3 and is identical to a sequence selected from SEQ ID No.1327-1768 and 1889 or comprises one or more amino acid residue substitutions relative to the sequence; and wherein f1, f2, f3 and f4 are framework residues.

[0044] One embodiment provides a method for treating or ameliorating a proliferative disease or a tumor disease, comprising administering a DLL3-targeting tri-specific protein according to the present disclosure to a subject in need thereof.

[0045] In one embodiment, a DLL3 targeting trispecific protein is provided, wherein the protein comprises (a) a first domain (A) that can specifically bind to human CD3; (b) a second domain (B) that is a half-life extension domain; and (c) a third domain (C) that can specifically bind to a DLL3 protein. In some embodiments, the domains are connected 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 parent molecule that specifically binds to the DLL3 protein. In some embodiments, the affinity matured binding molecules are derived from a parent molecule that specifically binds to DLL3 after an affinity maturation round. In some embodiments, the affinity maturation round comprises 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 parent molecule. In some embodiments, the phage display library expresses one or more molecules derived from the parent molecule. In some embodiments, the affinity matured binding molecules are selected from one or more molecules derived from the parent molecule. In some embodiments, the binding affinity of the affinity matured DLL3 binding molecules to the DLL3 protein is greater than the binding affinity of the parent molecule to the DLL3 protein. In some embodiments, the binding affinity of the affinity matured DLL3 binding molecules to the DLL3 protein is about 2 times to about 50 times greater than the binding affinity of the parent molecule to the DLL3 protein. In some embodiments, the affinity matured DLL3 binding molecule has a binding affinity for the DLL3 protein that is about 3 times greater than the binding affinity of the parent 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 Nos. 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.

[0046] In some embodiments, the 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, the 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, the CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889, or one or more substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889. In some embodiments, the 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, the CDR2 comprises a sequence selected from SEQ ID No. 937-970, or one or more amino acid substitutions relative to a sequence selected from SEQ ID No. 937-970. In some embodiments, the CDR3 comprises a sequence selected from SEQ ID No. 1379-1412, or one or more amino acid substitutions relative to a sequence selected from SEQ ID No. 1379-1412.

[0047] In some embodiments, the third domain (C) comprises a sequence that is at least about 80% identical to a sequence selected from SEQ ID No.53-86. In some embodiments, the CDR1 comprises a sequence selected from SEQ ID No.529-809, or one or more amino acid substitutions relative to a sequence selected from SEQ ID No.529-809. In some embodiments, the CDR2 comprises a sequence having one or more amino acid substitutions relative to a sequence selected from SEQ ID No.971-1251. In some embodiments, the CDR3 comprises a sequence selected from SEQ ID No.1379-1412, or one or more amino acid substitutions relative to a sequence selected from 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 SEQ ID No.87-367. In some embodiments, the CDR1 comprises a sequence selected from SEQ ID No.810-884, or one or more amino acid substitutions relative to a sequence selected from SEQ ID No.810-884. In some embodiments, the CDR2 comprises a sequence selected from SEQ ID No. 1252-1326, or one or more amino acid substitutions relative to a sequence selected from SEQ ID No. 1252-1326. In some embodiments, the CDR3 comprises a sequence selected from SEQ ID No. 1692-1768, or one or more amino acid substitutions relative to a sequence selected from SEQ ID No. 1692-1768. In some embodiments, the third domain (C) comprises a sequence at least about 75% identical to a sequence selected from SEQ ID No. 368-442.

[0048] In one embodiment, a DLL3-targeting trispecific protein is provided, wherein the protein comprises (a) a first domain (A) that can specifically bind to human CD3; (b) a second domain (B) that is a half-life extension domain; and (c) a third domain (C) that can specifically bind 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, the CDR1 comprises the following sequence: GX1X2X3X4X5NX6X7X8. In some embodiments, the CDR2 comprises the following sequence: GJ1SJ2J3J4J5J6J7J8J9J 10 SJ 11 KJ 12(SEQ ID No.1895).

[0049] In some embodiments, the CDR3 comprises the following sequence:

[0050] Z1Z2Z3Z4Z5Z6Z7Z8Z9Z 10 Z 11 In some embodiments:

[0051] X1 is A, E, F, G, I, K, L, N, Q, R, S, T, V or Y;

[0052] X2 is A, G, I, K, P, R, S, T or V;

[0053] X3 is A, D, F, K, L, N, P, Q, R, S, T or Y;

[0054] X4 is A, D, F, H, I, K, L, M, N, P, R, S, T, V or Y;

[0055] X5 is F, I, K, L, M, N, R, S, T or V;

[0056] X6 is A or G;

[0057] X7 is F, I, L, M, T, V or Y; and

[0058] X8 is A or G.

[0059] In some embodiments:

[0060] J1 is I or V;

[0061] J2 is A, K, P, R or S;

[0062] J3 is D or N;

[0063] J4 is D, E, G, K, N, Q, R, S, T or Y;

[0064] J5 is S or T;

[0065] J6 is A, E, F, H, I, K, L, N, Q, R, S, T, V or Y;

[0066] J7 is A, I, L, M, V or Y;

[0067] J 8为 D, F, H, I, L, N, S, T, V or Y;

[0068] J9 is A, D, E, F, G, I, K, L, N, Q, R, S, T, V or Y;

[0069] J 10is A, D, E, G, K, Q, S or V;

[0070] J 11 is A or V; and

[0071] J 12 It is G or V.

[0072] In some embodiments:

[0073] Z1 is F or Y;

[0074] Z2 is G, H, I, K, N, R, S or T;

[0075] Z3 is A, F, H, I, K, L, M, N, P, Q, R, S, T or Y;

[0076] Z4 is A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V or Y;

[0077] Z5 is A, C, D, E, G, H, I, K, L, N, P, Q, R, S, T, W or Y;

[0078] Z6 is G, K, L, R or T;

[0079] Z7 is A, G, H, L, Q, R, S, T, V or Y;

[0080] Z 8为 A, D, E, G, H, P, Q, S, T, W or Y;

[0081] Z9 is A, G, I, K, L, M, N, Q, R, S, T, V or Y;

[0082] Z 10 is A, G, K, P, R, S, T or V; and

[0083] Z 11 A, F, S or Y.

[0084] In one embodiment, a DLL3-targeting trispecific protein is provided, wherein the protein comprises (a) a first domain (A) that can specifically bind to human CD3; (b) a second domain (B) that is a half-life extension domain; and (c) a third domain (C) that can specifically bind 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 the CDR1 comprises the following sequence:

[0085] X1X2X3X4X5X6X7SX8A, wherein the CDR2 comprises the following sequence:

[0086] GJ1SJ2J3GJ4J5J6YJ7J8SVKG (SEQ ID No. 1894), and the CDR3 comprises the following sequence: Z1Z2Z3Z4Z5Z6Z7Z8Z9.

[0087] In some embodiments:

[0088] X1 is A, D, E, F, G, H, K, L, M, N, Q, R, S, V, W or Y;

[0089] X2 is D, E, H, K, M, P, R, S, T or Y;

[0090] X3 is A, D, G, H, K, N, P, Q, R, S, T, V or Y;

[0091] X4 is K, S or V;

[0092] X5 is A, F, G, H, K, L, M, N, Q, R, S, T, V, W or Y;

[0093] X6 is D, F, H, I, K, L, M, N, Q, R, S, V or Y;

[0094] X7 is L or M; and

[0095] X8 is I, L, M, S, T or V.

[0096] In some embodiments:

[0097] J1 is I or V;

[0098] J2 is A, D, E, G, H, I, K, L, N, P, Q, R, S, T, V or Y;

[0099] J3 is A, D, E, G, H, N, R or T;

[0100] J4 is H, P, R or S;

[0101] J5 is A, H, I, K, M, N, Q, R, T or V;

[0102] J6 is A, D, G, H, I, L, M, N, S, T, V or Y;

[0103] J7 is A, F, I, L, M, R, S, T, V or Y;

[0104] J8 is A, D, E, G, H, K, L, N, R, S or V;

[0105] In some embodiments:

[0106] Z1 is L or Y;

[0107] Z2 is D, E, G, H, K, N, Q, R, S, T, V or Y;

[0108] Z3 is Q or W;

[0109] Z4 is A, D, E, G, H, I, K, L, M, P, R, S, T or V;

[0110] Z5 is A, D, E, G, N, R, S, T or Y;

[0111] Z6 is A, P, R or S;

[0112] Z7 is A, D, F, G, H, L, M, N, Q, R, S, T, V or Y;

[0113] Z8 is A, G, I, K, P, Q, R, S or T; and

[0114] Z9 is F, H or Y.

[0115] 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, a VHH domain, a scFv, a VH domain, a VL domain, a Fab, a Fab', a non-Ig domain, a ligand, a knottin, or a small molecule entity that specifically binds to DLL3. In some embodiments, the third domain (C) comprises the single domain antibody. In some embodiments, the second domain (B) binds to a bulky serum protein. In some embodiments, the second domain (B) comprises a single domain antibody, a VHH domain, a scFv, a VH domain, a VL domain, a Fab, a Fab', a non-Ig domain, a ligand, a knottin, or a small molecule entity that specifically binds to the bulky serum protein. In some embodiments, the second domain (B) comprises a single domain antibody that specifically binds to the bulky serum protein. In some embodiments, the bulk serum protein comprises albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, pentamer IgM or light chain without Igκ. In some embodiments, the bulk serum protein comprises albumin. In some embodiments, the second domain (B) comprises a sequence at least about 75% identical to a sequence selected from SEQ ID No.1769-1778. In some embodiments, the second domain (B) comprises a sequence at least about 75% identical to SEQ ID No.1774.

[0116] In some embodiments, the first domain (A) comprises a single domain antibody, a VHH domain, a scFv, a VH domain, a VL domain, a Fab, a Fab', a non-Ig domain, a ligand, a knottin or a small molecule entity that specifically binds to CD3. In some embodiments, the first domain (A) comprises a sequence selected from SEQ ID No. 1793-1807. In some embodiments, the third domain (C) comprises the following formula:

[0117] f1-r1-f2-r2-f3-r3-f4

[0118] wherein r1 is identical to a sequence selected from SEQ ID No.443-884 and 1887 or comprises one or more amino acid residue substitutions relative to the sequence; r2 is identical to a sequence selected from SEQ ID No.885-1326 and 1888 or comprises one or more amino acid residue substitutions relative to the sequence; and r3 is identical to a sequence selected from SEQ ID No.1327-1768 and 1889 or comprises one or more amino acid residue substitutions relative to the sequence; and wherein f1, f2, f3 and f4 are framework residues. In some embodiments, the third domain (C) comprises a sequence at least 75% identical to a sequence selected from SEQ ID No.1-442 and 1886. In some embodiments, domains C and B are connected by a linker L1, and domains B and A are connected by a linker L2. In some embodiments, linkers L1 and L2 are each independently selected from (GS) n (SEQ ID No.1809), (GGS) n (SEQ ID No.1810), (GGGS) n (SEQ ID No.1811), (GGSG) n (SEQ ID No.1812), (GGSGG) n (SEQ ID No. 1813) or (GGGGS) n(SEQ ID No.1814), wherein 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 kDa to about 75 kDa. In some embodiments, the protein is less than about 60 kDa. In some embodiments, the protein has an elimination half-life of at least about 50 hours. In some embodiments, the protein has an elimination half-life of at least about 100 hours. In some embodiments, the protein has increased tissue penetration for the same DLL3 compared to IgG. In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH.

[0119] One embodiment provides a pharmaceutical composition comprising (i) a DLL3 targeting 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 targeting trispecific binding protein according to any one of the above embodiments, the method comprising: i) providing a DLL3 protein or a fragment thereof; ii) exposing a recombinant library of DLL3 binding proteins to the DLL3 protein or a fragment thereof; iii) selecting a DLL3 binding protein that specifically binds to the oligomer or a derivative thereof from the library; and (iv) preparing a DLL3 targeting trispecific protein using the DLL3 binding protein identified in step (iii). 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 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.

[0120] One embodiment provides a method for producing a DLL3-targeting tri-specific protein according to any one of the above embodiments, the method comprising culturing a host transformed or transfected with a vector under conditions allowing expression of the DLL3-targeting tri-specific protein, the vector comprising a nucleic acid sequence encoding the DLL3 tri-specific protein according to any one of the above embodiments, and recovering and purifying the produced protein from the culture.

[0121] One embodiment provides a DLL3 binding protein comprising the formula:

[0122] f1-r1-f2-r2-f3-r3-f4

[0123] wherein r1 is a complementarity 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 Nos. 443-884 and 1887; r2 is a 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 Nos. 885-1326 and 1888; and r3 is a 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 Nos. 1327-1768 and 1889; and wherein f1, f2, f3 and f4 are framework residues. In some embodiments, the DLL3 binding protein comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886. In some embodiments, the DLL3 binding protein comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-52. In some embodiments, the DLL3 binding protein comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 53-86. In some embodiments, the DLL3 binding protein comprises a sequence that is at least about 75% identical to a sequence selected from 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 SEQ ID No. 368-442. One embodiment provides a DLL3 binding protein derived from a parent DLL3 binding protein comprising the sequence of SEQ ID No. 68. One embodiment provides a DLL3 binding protein derived from a parent DLL3 binding protein comprising the sequence of SEQ ID No. 75, or comprises the sequence of SEQ ID No. 75.

[0124] One embodiment provides a method of preparing a DLL3 binding protein according to the present disclosure, the method comprising: i) providing a DLL3 protein or a fragment thereof; ii) exposing a recombinant library of DLL3 binding proteins to the DLL3 protein or a fragment thereof; iii) selecting a DLL3 binding protein from the library 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 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 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.

[0125] One embodiment provides a method for producing a DLL3 binding protein according to the present disclosure, the method 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 allowing expression of the DLL3 binding protein, and recovering and purifying the produced protein from the culture.

[0126] One embodiment provides a method for treating or ameliorating a proliferative disease or a neoplastic disease, comprising administering to a subject in need thereof 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 provided herein. In some embodiments, the subject is a human. In some embodiments, the method further comprises administering a pharmaceutical agent in combination with a DLL3-targeted trispecific protein according to the above embodiments, a DLL3-binding protein according to the present disclosure, or a pharmaceutical composition 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 comprises a solid tumor disease. In some embodiments, the solid tumor disease comprises lung cancer, gastric cancer, ovarian cancer, or triple-negative breast cancer. In some embodiments, the solid tumor disease is metastatic.

[0127] One embodiment provides a method for treating or ameliorating a proliferative disease or a neoplastic disease, comprising administering a DLL3-targeting trispecific protein comprising a DLL3-binding domain comprising a sequence selected from SEQ ID No. 1-442 and 1886, or a DLL3-binding protein comprising a sequence selected from SEQ ID No. 1-442 and 1886. In some embodiments, the DLL3-targeting trispecific protein or DLL3-binding protein selectively binds to tumor cells expressing DLL3. In some embodiments, the DLL3-targeting trispecific protein directs T cells to kill tumor cells expressing DLL3. In some embodiments, the neoplastic disease comprises a solid tumor disease. In some embodiments, the solid tumor disease comprises lung cancer, gastric cancer, ovarian cancer, or triple-negative breast cancer. In some embodiments, the solid tumor disease is metastatic.

[0128] One embodiment provides a method for treating or ameliorating a proliferative disease or a neoplastic disease, comprising administering a DLL3 binding domain comprising a sequence as shown in SEQ ID No. 68 or 75, or a DLL3 binding protein comprising a sequence as shown in SEQ ID No. 68 or 75. In some embodiments, the dosage of the DLL3 targeting trispecific protein or DLL3 binding protein is 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 other week. In some embodiments, the protein is administered once every three weeks.

[0129] In one embodiment, a DLL3 binding protein is provided, which comprises the amino acid sequence shown in SEQ ID No.1890 or SEQ ID No.1891.

[0130] One embodiment provides a DLL3-targeting trispecific protein, wherein the protein comprises (a) a first domain (A), which is a single-chain variable fragment that can specifically bind to human CD3; (b) a second domain (B), which is a single-domain antibody that can specifically bind to human serum albumin; and (c) a third domain (C), which is a single-domain antibody that can specifically bind to DLL3 protein, wherein the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from SEQ ID No. 1-442 and 1886.

[0131] In some embodiments, the domains are connected 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 through 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, H2N-(C)-L1-(B)-L2-(A)-COOH or H2N-(C)-L1-(A)-L2-(B)-COOH are connected in sequence.

[0132] 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 DLL3 binding molecule has a binding affinity for the DLL3 protein that is about 2 to about 50 times greater than the binding affinity of the parent molecule for the DLL3 protein.

[0133] In some embodiments, the third domain (C) comprises a sequence selected from SEQ ID No.1-442 and 1886. In some embodiments, the third domain (C) comprises a sequence selected from SEQ ID No.1-52. In some embodiments, the third domain (C) comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises a sequence selected from SEQ ID No.443-884 and 1887, wherein the CDR2 comprises a sequence selected from SEQ ID No.885-1326 and 1888, and wherein the CDR3 comprises a sequence selected from SEQ ID No.1327-1768 and 1889. In some embodiments, the CDR1 comprises a sequence selected from SEQ ID No.495-528. In some embodiments, the CDR2 comprises a sequence selected from SEQ ID No.937-970. In some embodiments, the CDR3 comprises a sequence selected from SEQ ID No.1379-1412. In some embodiments, the third domain (C) comprises a sequence selected from SEQ ID No. 53-86.

[0134] In some embodiments, the third domain (C) comprises CDR1, CDR2 and CDR3, and wherein the CDR1 comprises a sequence selected from SEQ ID No.529-809. In some embodiments, the CDR2 comprises a sequence selected from SEQ ID No.971-1251. In some embodiments, the CDR3 comprises a sequence selected from SEQ ID No.1379-1412. In some embodiments, the third domain (C) comprises a sequence selected from SEQ ID No.87-367. In some embodiments, the third domain (C) comprises CDR1, CDR2 and CDR3, and wherein the CDR1 comprises a sequence selected from SEQ ID No.810-884. In some embodiments, the CDR2 comprises a sequence selected from SEQ ID No.1252-1326. In some embodiments, the CDR3 comprises a sequence selected from SEQ ID No.1692-1768. In some embodiments, the third domain (C) comprises a sequence selected from SEQ ID No.368-442. In some embodiments, the first domain (A) comprises a sequence selected from SEQ ID Nos. 1793-1807 and 1897-1898.

[0135] In some embodiments, the second domain (B) comprises a sequence selected from SEQ ID No. 1769-1778. In some embodiments, linkers L1 and L2 are each independently selected from (GS) n (SEQ ID No.1809), (GGS) n (SEQ ID No. 1810), (GGGS) n (SEQ ID No.1811), (GGSG) n (SEQ ID No.1812), (GGSGG) n (SEQ ID No. 1813) or (GGGGS) n (SEQ ID No.1814), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the domains are connected in the order of H2N-(A)-(B)-(C)-COOH or through 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 of GGGGSGGGS (SEQ ID No.1808). In some embodiments, the DLL3 targeting trispecific protein comprises the sequence of SEQ ID No.1890 or SEQ ID No.1891.

[0136] One embodiment provides a DLL3-targeting trispecific protein, wherein the protein comprises (a) a first domain (A), which is a single-chain variable fragment that can specifically bind to human CD3; (b) a second domain (B), which is a single-domain antibody that can specifically bind to human serum albumin; and (c) a third domain (C), which is a single-domain antibody that can specifically bind 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. 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.

[0137] One embodiment provides a DLL3 binding protein comprising the formula:

[0138] f1-r1-f2-r2-f3-r3-f4

[0139] wherein r1 is a complementarity determining region 1 (CDR1) and is identical to a sequence selected from SEQ ID Nos. 443-884 and 1887 or comprises one or more amino acid residue substitutions relative to the sequence; r2 is a CDR2 and is identical to a sequence selected from SEQ ID Nos. 885-1326 and 1888 or comprises one or more amino acid residue substitutions relative to the sequence; and r3 is a CDR3 and is identical to a sequence selected from SEQ ID Nos. 1327-1768 and 1889 or comprises one or more amino acid residue substitutions relative to the sequence; and wherein f1, f2, f3 and f4 are framework residues. In some embodiments, the CDR1 comprises a sequence selected from SEQ ID Nos. 443-884 and 1887, the CDR2 comprises a sequence selected from SEQ ID Nos. 885-1326 and 1888, and the CDR3 comprises a sequence selected from SEQ ID Nos. 1327-1768 and 1889.

[0140] One embodiment provides a DLL3-targeting trispecific protein, wherein the protein comprises (a) a first domain (A), which is a single-chain variable fragment that can specifically bind to human CD3; (b) a second domain (B), which is a single-domain antibody that can specifically bind to human serum albumin; and (c) a third domain (C), which is a single-domain antibody that can specifically bind to the DLL3 protein, wherein the third domain (C) comprises a CDR1 having an amino acid sequence of SEQ ID No.874, a CDR2 having an amino acid sequence of SEQ ID No.1316, and a CDR3 having an amino acid sequence of SEQ ID No.1758.

[0141] One embodiment provides a DLL3 targeting trispecific protein, wherein the protein comprises (a) a first domain (A), which is a single-chain variable fragment that can specifically bind to human CD3; (b) a second domain (B), which is a single-domain antibody that can specifically bind to human serum albumin; and (c) a third domain (C), which is a single-domain antibody that can specifically bind to DLL3, wherein the third domain comprises a CDR1 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 851, 867, 871, 872, 873, 874 and 1887; a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 1293, 1309, 1313, 1314, 1315, 1316 and 1888; and a CDR3 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 1735, 1751, 1755, 1756, 1757, 1758 and 1889. In some embodiments, the third domain (C) comprises a sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID Nos. 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 Nos. 408, 425, 432, 430, 431 and 1886.

[0142] In some embodiments, the domains are connected 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 through 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, H2N-(C)-L1-(B)-L2-(A)-COOH or H2N-(C)-L1-(A)-L2-(B)-COOH are connected in sequence.

[0143] In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH or in the order of H2N-(A)-L1-(B)-L2-(C)-COOH through linkers L1 and L2. In some embodiments, the domains are linked in the order of H2N-(C)-(B)-(A)-COOH or in the order of H2N-(C)-L1-(B)-L2-(A)-COOH through linkers L1 and L2. 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.

[0144] In some embodiments, the affinity matured DLL3 binding molecule has a binding affinity for the DLL3 protein that is about 2 to about 50 times 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.

[0145] In some embodiments, linkers L1 and L2 are each independently selected from (GS) n (SEQ ID No.1809), (GGS) n (SEQ ID No.1810), (GGGS) n (SEQ ID No.1811), (GGSG) n (SEQ ID No.1812), (GGSGG) n (SEQ ID No.1813) or (GGGGS) n(SEQ ID No. 1814), wherein 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).

[0146] 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 targeting tri-specific protein comprises a 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 a sequence of SEQ ID No. 1893.

[0147] One embodiment provides a DLL3 binding protein comprising a CDR1 having an amino acid sequence of SEQ ID No. 874, a CDR2 having an amino acid sequence of SEQ ID No. 1316, and a CDR3 having an 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.

[0148] One embodiment provides a DLL3 binding protein comprising a CDR1 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 851, 867, 871, 872, 873, 874, and 1887; a CDR2 having an amino acid sequence selected from the group consisting of SEQ ID Nos. 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. 1 and SEQ ID Nos. 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 Nos. 408, 425, 432, 430, 431, and 1886.

[0149] One embodiment provides a method for treating or ameliorating a disease, comprising administering an effective amount of a DLL3-targeting tri-specific protein according to any one of the above embodiments.

[0150] Incorporation by reference

[0151] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention will be better understood by referring to the following detailed description of illustrative embodiments utilizing the principles of the present invention and the accompanying drawings, in which:

[0153] Figure 1 The various domains of an exemplary DLL3-targeting tri-specific protein of the present disclosure are shown.

[0154] Figure 2 Shown are the results of a T cell-dependent cellular cytotoxicity (TDCC) assay on DMS-153 cells using an exemplary DLL3-targeting trispecific protein comprising the DLL3-binding domains DH18, DH11, DH67, and DH56 of the present disclosure.

[0155] Figure 3 Shown are the results of a TDCC assay on DMS-153 cells using an exemplary DLL3-targeting trispecific protein comprising exemplary DLL3-binding domains DH2, DH43, DH10, and DH6 of the disclosure.

[0156] Figure 4 Shown are the results of a TDCC assay on DMS-153 cells using an exemplary DLL3-targeting trispecific protein comprising exemplary DLL3-binding domains DH82, DH23, DH89, and DH17 of the disclosure.

[0157] Figure 5 Shown are the results of a TDCC assay on DMS-153 cells using an exemplary DLL3-targeting trispecific protein comprising exemplary DLL3 binding domains DH83, DH12, DH61, and DH29 of the disclosure.

[0158] Figure 6 Shown are the results of a TDCC assay on DMS-153 cells using an exemplary DLL3-targeting trispecific protein comprising exemplary DLL3-binding domains DH58 and DH70 of the disclosure, as well as a control trispecific protein.

[0159] Figure 7 Shown are the results of a TDCC assay on DMS-153 cells using an exemplary affinity matured DLL3 targeting trispecific protein comprising exemplary DLL3 targeting domains 1A011, 2E05, 1H012, 2E02 and 1C03 of the present disclosure.

[0160] Figure 8 Shown are the results of a TDCC assay on DMS-153 cells using an exemplary affinity matured DLL3 binding trispecific protein comprising exemplary DLL3 targeting domains 2E010, 2E01, 2H02, 2A04 and 2F11 of the disclosure.

[0161] Fig. 9 Shown are the results of a TDCC assay on DMS-153 cells using an exemplary affinity matured DLL3 binding trispecific protein comprising exemplary DLL3 targeting domains 2E011, 3C04, 4H04, 4H011, and 4D09 of the disclosure.

[0162] Fig.10 Shown are the results of a TDCC assay on DMS-153 cells using an exemplary affinity matured DLL3 binding trispecific protein comprising exemplary DLL3 targeting domains 4B07, 4E02, 4C06, 3H011 and 3D07 of the disclosure.

[0163] Fig.11 Shown are the results of a TDCC assay on DMS-153 cells using exemplary affinity-matured DLL3-targeting tri-specific proteins comprising exemplary DLL3 binding domains 3H06 and 4B011 of the disclosure and parental DLL binding domains DH43, DH6, as well as a control tri-specific protein.

[0164] Fig.12 Shown are the results of a TDCC assay on DMS-153 cells using exemplary purified, affinity matured, CHO expressed DLL3 binding trispecific proteins comprising exemplary DLL3 targeting domains 2E05-M106Y, 2E05-M106Q, 4D09-M34L and 4H11-M34L of the disclosure.

[0165] Fig.13 The use of an exemplary DLL3 binding domain comprising 1A011 of the present disclosure (in Fig.13 Marked as 1A11), 1H012 (on Fig.13 Results of TDCC assay on DMS-153 cells for exemplary purified, affinity-matured, CHO-expressed DLL3-targeting trispecific proteins labeled 1H12), 2E02, and 2E05.

[0166] Fig.14 Shown are the results of a TDCC assay on DMS-153 cells using exemplary purified, affinity matured, CHO expressed DLL3-targeting trispecific proteins comprising exemplary DLL3 binding domains 2H02, 3C04, 4D09 and 4H11 of the disclosure.

[0167] Fig.15 Shown are the results of a TDCC assay on DMS-153 cells using exemplary purified DLL3-targeted trispecific proteins comprising parental exemplary DLL3 binding domains DH43 and DH6, and a control trispecific protein targeting GFP.

[0168] Fig.16 Shown are the results of a TDCC assay on DMS-153 cells using an exemplary DLL3-targeting trispecific protein comprising an exemplary DLL3-binding domain of the disclosure from the second round of affinity maturation.

[0169] Fig.17 An image of a 10-20% TRIS glycine SDS-PAGE loaded with 2.4 micrograms of non-reduced protein per lane and stained with Coomassie is shown. Lane numbers are indicated by numbers at the top of the gel image and the migration of molecular weight standards is indicated by numbers (in kilodaltons) to the right of the gel image. Gel loading: lane 1 is empty, lane 2 contains molecular weight standard, lane 3 is empty, lane 4 contains anti-DLL3 trispecific protein with DLL3 binding domain 51G2, lane 5 contains anti-DLL3 trispecific protein with DLL3 binding domain 51G10, lane 6 contains anti-DLL3 trispecific protein with DLL3 binding domain 51H5, lane 7 contains anti-DLL3 trispecific protein with DLL3 binding domain 51X5, lane 8 contains anti-DLL3 trispecific protein with DLL3 binding domain 52B1, lane 9 contains anti-DLL3 trispecific protein with DLL3 binding domain 52C4, lane 10 contains anti-DLL3 trispecific protein with DLL3 binding domain 52D4, lane 11 contains anti-DLL3 trispecific protein with DLL3 binding domain 51A2, lane 12 contains anti-DLL3 trispecific protein with DLL3 binding domain 51A5, lane 13 contains anti-DLL3 trispecific protein with DLL3 binding domain 51F3, lane 14 is empty, lane 15 is empty.

[0170] Fig.18 Shown are the results of a TDCC assay on DMS-53 cells using exemplary purified, affinity matured, CHO expressed DLL3-targeting tri-specific proteins comprising exemplary DLL3 binding domains 51G2, 51G10, 51H5, 51X5, 52B1, 52C4, 52D4, 51A2 and the parental DLL3 binding domain DH6 of the present disclosure, as well as a control tri-specific protein.

[0171] Fig.19Shown are the results of a TDCC assay on DMS-153 cells using exemplary purified, affinity matured, CHO expressed DLL3-targeting tri-specific proteins of the present disclosure comprising exemplary DLL3 binding domains 51G2, 51G10, 51H5, 51X5, 52B1, 52C4, 52D4, 51A2 and the parental DLL3 binding domain DH6 of the present disclosure, as well as a control binding tri-specific protein targeting GFP.

[0172] Fig. 20 A schematic diagram of a DLL3-targeting trispecific protein is provided, which comprises an exemplary DLL3 binding protein (DLL3 binder) of the present disclosure, a CD3 binding domain (anti-CD3 epsilon scFv), and an albumin binding (anti-ALB) domain in an anti-DLL3:anti-ALB:anti-CD3 orientation (TAC orientation).

[0173] Fig.21 A schematic diagram of a DLL3-targeting trispecific protein is provided, which comprises an exemplary DLL3 binding protein (DLL3 binder) of the present disclosure, a CD3 binding domain (anti-CD3 epsilon scFv), and an albumin binding (anti-ALB) domain in an anti-CD3:anti-ALB:anti-DLL3 orientation (CAT orientation).

[0174] Fig. 22 Shown are the results of a T cell-dependent cytotoxicity (TDCC) assay on NCI-H2171 cells using an exemplary DLL3 trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration or an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) or bovine serum albumin (BSA).

[0175] Fig.23 Shown are the results of a T cell-dependent cytotoxicity (TDCC) assay on DMS-79 cells using an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration or an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence or absence of human serum albumin (HSA).

[0176] Fig.24Shown are the results of a T cell-dependent cytotoxicity (TDCC) assay on SHP77 cells using an exemplary DLL3 trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration or an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) or bovine serum albumin (BSA).

[0177] Fig.25 Shown are the results of a T cell-dependent cytotoxicity (TDCC) assay on WM2664 cells using an exemplary DLL3 trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration or an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) or bovine serum albumin (BSA).

[0178] Fig.26 Depicted is binding of an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration to human T cells from four different donors compared to a control containing only a secondary antibody or cells without any antibody or trispecific molecule.

[0179] Fig. 27 Depicted is binding of an exemplary DLL3 targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration to human T cells from four different donors compared to a control containing only a secondary antibody or cells without any antibody or trispecific molecule.

[0180] Fig.28 Depicted is the binding of an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration to the human DLL3-expressing cell lines NCI-H82 (upper left), SHP77 (upper right), DMS53 (lower left), or NCI-H2171 (lower right).

[0181] Fig.29 Depicted is binding of an exemplary DLL3 targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration to the human DLL3 expressing cell lines NCI-H82 (upper left), SHP77 (upper right), DMS53 (lower left), or NCI-H2171 (lower right).

[0182] Fig.30 Shown are the results of a TDCC assay using an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration on NCI-H82 cells, tested in the presence of human serum albumin (HSA) using T cells from four different donors.

[0183] Fig.31 Shown are the results of a TDCC assay using an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration on SHP77 cells, tested in the presence of human serum albumin (HSA) using T cells from four different donors.

[0184] Fig.32 Shown are the results of a TDCC assay using an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration on DMS53 cells, tested in the presence of human serum albumin (HSA) using T cells from four different donors.

[0185] Fig.33 Shown are the results of a TDCC assay using an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration on NCI-H2171 cells, tested in the presence of human serum albumin (HSA) using T cells from four different donors.

[0186] Fig.34 Shown are the results of a TDCC assay using an exemplary DLL3 targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration on NCI-H82 cells, tested in the presence of human serum albumin (HSA) using T cells from four different donors.

[0187] Fig.35 Shown are the results of a TDCC assay using an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration on SHP77 cells, tested in the presence of human serum albumin (HSA) using T cells from four different donors.

[0188] Fig.36Shown are the results of a TDCC assay using an exemplary DLL3 targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration on DMS53 cells, tested in the presence of human serum albumin (HSA) using T cells from four different donors.

[0189] Fig.37 Shown are the results of a TDCC assay using an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration on NCI-H2171 cells, tested in the presence of human serum albumin (HSA) using T cells from four different donors.

[0190] Fig.38 Shown are flow cytometric measurements of CD69 expression on T cells co-cultured with NCI-H82 cells, in which an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration was titrated, using T cells from four different donors, tested in the presence of human serum albumin (HSA).

[0191] Fig.39 Shown are flow cytometric measurements of CD25 expression on T cells co-cultured with NCI-H82 cells, in which an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration was titrated, using T cells from four different donors, tested in the presence of human serum albumin (HSA).

[0192] Fig.40 Shown are flow cytometric measurements of CD69 expression on T cells co-cultured with DMS53 cells, in which an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration was titrated, using T cells from four different donors, tested in the presence of human serum albumin (HSA).

[0193] Fig.41 Shown are flow cytometric measurements of CD25 expression on T cells co-cultured with DMS53 cells, in which an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration was titrated and tested in the presence of human serum albumin (HSA).

[0194] Fig.42Shown are flow cytometric measurements of CD69 expression on T cells co-cultured with NCI-H82 cells, in which an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration was titrated, using T cells from four different donors, tested in the presence of human serum albumin (HSA).

[0195] Fig.43 Shown are flow cytometric measurements of CD25 expression on T cells co-cultured with NCI-H82 cells, in which an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration was titrated, using T cells from four different donors, tested in the presence of human serum albumin (HSA).

[0196] Fig.44 Shown are flow cytometric measurements of CD69 expression on T cells co-cultured with DMS53 cells, in which an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration was titrated, using T cells from four different donors, tested in the presence of human serum albumin (HSA).

[0197] Fig.45 Shown are flow cytometric measurements of CD25 expression on T cells co-cultured with DMS53 cells, in which an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration was titrated and tested in the presence of human serum albumin (HSA).

[0198] Fig.46 Shown are the results of IFNγ measurement in conditioned media of co-cultures of T cells and NCI-H82 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0199] Fig.47 Shown are the results of IFNγ measurement in conditioned media of co-cultures of T cells and SHP77 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0200] Fig.48 Shown are the results of IL-2 measurement in conditioned media from co-cultures of T cells and NCI-H82 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0201] Fig.49 Shown are the results of IL-2 measurement in conditioned media of co-cultures of T cells and SHP77 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0202] Fig.50 Shown are the results of TNFα measurement in conditioned media from co-cultures of T cells and NCI-H82 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0203] Fig.51 Shown are the results of TNFα measurement in conditioned media from co-cultures of T cells and SHP77 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0204] Fig.52 Shown are the results of IFNγ measurement in conditioned media of co-cultures of T cells and NCI-H82 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration, tested in the presence of human serum albumin (HSA).

[0205] Fig.53 Shown are the results of IFNγ measurement in conditioned media of co-cultures of T cells and SHP77 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration, tested in the presence of human serum albumin (HSA).

[0206] Fig.54 Shown are the results of IL-2 measurement in conditioned media of co-cultures of T cells and NCI-H82 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration, tested in the presence of human serum albumin (HSA).

[0207] Fig.55 Shown are the results of IL-2 measurement in conditioned media from co-cultures of T cells and SHP77 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration, tested in the presence of human serum albumin (HSA).

[0208] Fig.56 Shown are the results of TNFα measurement in conditioned media from co-cultures of T cells and NCI-H82 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration, tested in the presence of human serum albumin (HSA).

[0209] Fig.57 Shown are the results of TNFα measurement in conditioned media of co-cultures of T cells and SHP77 cells incubated with titrated exemplary DLL3-targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration, tested in the presence of human serum albumin (HSA).

[0210] Fig.58 Depicted is the ability of an exemplary DLL3-targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration or an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration to inhibit tumor growth in mice injected with a mixture of human T cells and NCI-H82 small cell lung cancer cells at doses of 20 μg / kg, 100 μg / kg, or 500 μg / kg.

[0211] Fig.59Depicted that an exemplary DLL3-targeting trispecific protein comprising the disclosed DLL3 binding domain 52D04 in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration was able to abrogate the growth of NCI-H82 xenograft tumors at 10 μg / kg and 100 μg / kg doses in mice injected with human T cells.

[0212] Fig.60 Depicted that an exemplary DLL3-targeting trispecific protein comprising the disclosed DLL3 binding domain 52D04 in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration was able to inhibit tumor growth at 10 μg / kg and 100 μg / kg doses in mice injected with a mixture of human T cells and SHP77 small cell lung cancer cells.

[0213] Fig.61 Depicted are pharmacokinetic profiles of exemplary DLL3-targeted trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in anti-CD3: anti-ALB: anti-DLL3 (CAT) configurations (ID Nos. 1 and 2) or anti-DLL3: anti-ALB: anti-CD3 (TAC) configurations (ID Nos. 3 and 4). The figure shows serum levels of the DLL3-targeted trispecific protein at different time points after injection into cynomolgus monkeys at 0.3 mg / kg.

[0214] Fig.62 Depicted is the pharmacokinetic profile of an exemplary DLL3-targeted trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3: anti-ALB: anti-DLL3 (CAT) configuration. The graph shows different time points after injection into cynomolgus monkeys at 1 mg / kg or 10 mg / kg.

[0215] Fig.63 Depicted are transient cytokine increases following first administration of exemplary DLL3-binding TriTAC molecules of the present disclosure at 1 mg / kg and 10 mg / kg doses or vehicle control. The top graph shows a transient increase in IFNγ, the second graph shows a transient increase in IL-6, and the third graph shows a transient increase in IL-10.

[0216] Fig.64 Shown are the results of a TDCC assay performed on DMS53 cells using an exemplary DLL3-targeted trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-CD3:anti-ALB:anti-DLL3(CAT) configuration, measured in the presence of 8.4% cynomolgus monkey serum using freshly thawed protein or using protein present in a cynomolgus monkey serum sample collected 168 hours after administration of 10 mg / kg of the DLL3-targeted trispecific protein. DETAILED DESCRIPTION

[0217] In some embodiments, described herein are multispecific (e.g., trispecific) proteins that specifically bind to delta-like ligand 3 (DLL3) and contain it, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for preparing such proteins. Also provided are methods for preventing and / or treating diseases, conditions, and disorders using at least one of the disclosed DLL3 binding proteins or DLL3-targeted trispecific proteins containing it. The DLL3-targeted trispecific protein is capable of specifically binding to DLL3 as well as CD3, and has a half-life extension domain, such as a domain capable of specifically binding to human albumin (ALB). Figure 1 A non-limiting example of a trispecific DLL3 binding protein is depicted.In some embodiments, the DLL3 targeting trispecific protein comprises an antibody, such as a trispecific antibody.

[0218] Some definitions

[0219] "Antibody" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Human light chains comprise a variable domain (VL) and a constant domain (CL), wherein the constant domain can be readily classified as either κ or λ based on amino acid sequence and locus. Each heavy chain comprises a variable domain (VH) and a constant region, which in the case of IgG, IgA and IgD comprises three domains, referred to as CH1, CH2 and CH3, respectively (IgM and IgE have a fourth domain, CH4). In the IgG, IgA and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a proline and cysteine-rich segment of variable length (usually about 10 to about 60 amino acids in IgG). The variable domains in both the light and heavy chains are connected to the constant domains by a "J" region of about 12 or more amino acids, and the heavy chain also has a "D" region of about 10 additional amino acids. Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine ​​residues. There are two types of natural disulfide bridges or disulfide bonds in immunoglobulin molecules: interchain and intrachain disulfide bonds. The position and number of interchain disulfide bonds vary depending on the class and type of immunoglobulin. Interchain disulfide bonds are located on the surface of immunoglobulins, are solvent accessible, and are generally relatively easy to reduce. In the human IgG1 isotype, there are four interchain disulfide bonds, one from each heavy chain to the light chain, and two between the heavy chains. Interchain disulfide bonds are not required for chain association. It is well known that the cysteine-rich IgG1 hinge region of the heavy chain is usually maintained as consisting of three parts: an upper hinge, a core hinge, and a lower hinge. Those skilled in the art will appreciate that the IgG1 hinge region contains cysteines in the heavy chain that form interchain disulfide bonds (two heavy / heavy, two heavy / light) that provide structural flexibility that facilitates Fab movement. The interchain disulfide bond between the light and heavy chains of IgG1 is formed between C214 of the kappa or lambda light chain and C220 in the hinge region on the heavy chain. The interchain disulfide bond between the heavy chains is located at positions C226 and C229 (all numbered according to the EU index, according to Kabat et al., below).

[0220] As used herein, the term "antibody" includes polyclonal antibodies, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, 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, including muteins and variants thereof, immunospecific antibody fragments, such as Fd, Fab, F(ab')2, F(ab') fragments, single-chain fragments (e.g., ScFv and ScFvFc), disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies, such as sdAb (VH, VL or VHH domains); and derivatives thereof, including Fc fusions and other modifications, as well as any other immunoreactive molecules, as long as they contain a domain having a binding site that preferentially associates or binds to a DLL3 protein. In addition, unless otherwise indicated by contextual constraints, the term also includes antibodies of all classes (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 different antibody classes are generally represented by the corresponding lowercase Greek letters α, δ, ε, γ, and μ, respectively. The light chains of antibodies from any vertebrate species can be classified into one of two distinct types (called κ (kappa) and λ (lambda)) based on the amino acid sequence of their constant domains.

[0221] In some embodiments, the DLL3 binding domain of the DLL3 targeting trispecific protein of the present disclosure comprises a heavy chain-only antibody, such as a VH or VHH domain. In some cases, the DLL3 binding protein comprises a heavy chain-only antibody, which is an engineered VH domain. In some instances, the engineered human VH domain is generated by panning a phage display library. In some embodiments, the DLL3 binding domain of the DLL3 targeting trispecific protein of the present disclosure comprises a VHH. The term "VHH" as used herein refers to a single-chain antibody binding domain that does not contain a light chain. In some cases, the VHH is derived from an antibody of a type that naturally lacks a light chain that can be found in camelids or cartilaginous fish, or from a synthetic and non-immune VHH that can be constructed accordingly. Each heavy chain comprises a variable region encoded by V-, D-, and J-exons. In some cases, the VHH is a natural VHH, such as a camelid-derived VHH, or a recombinant protein comprising a heavy chain variable domain. In some embodiments, the VHH is derived from a species selected from camels, llamas, vicugnas, guanacos, and cartilaginous fish (such as, but not limited to, sharks). In another embodiment, the VHH is derived from an alpaca (such as, but not limited to, a Huacaya Alpaca or a Surialpaca).

[0222] As used herein, "variable region" or "variable domain" refers to the following situation: some parts of the variable domain are widely different in sequence between antibodies, and are used in the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed throughout the variable domain of the antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light chain (VL) and heavy chain (VH) variable domains. The more highly conserved parts of the variable domain are called frameworks (FRs). The variable domains of natural heavy and light chains each contain four FR regions, which mainly adopt a β-folded configuration, connected by three CDRs, which form loops connecting the β-folded structure, and in some cases form a part of the β-folded structure. The CDRs in each chain are kept together in close proximity by the FR region, and together with the CDRs from the other chain, help to form 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)). Although the constant domain is not directly involved in the binding of the antibody to the antigen, it exhibits various effector functions, such as the antibody's participation in the antibody-dependent cellular toxicity of the antibody. In some cases, the scFv fragment (variable for single-chain fragments) obtained by genetic engineering associates the VH and VL regions of the antibody in a single polypeptide chain, and the two regions are separated by a peptide linker.

[0223] In some embodiments of the present disclosure, the DLL3 binding domain, such as the DLL3 binding domain of a DLL3 targeting trispecific protein, comprises a single domain antibody, such as a heavy chain-only antibody, such as a VH or VHH domain, and comprises three CDRs. In some embodiments, such heavy chain-only antibodies bind DLL3 as a monomer, and their optimal binding affinity is independent of dimerization with a VL (light chain variable) region. In some embodiments of the present disclosure, the CD3 binding domain of the DLL3 targeting trispecific protein comprises a scFv. In some embodiments of the present disclosure, the albumin binding domain of the DLL3 targeting trispecific protein comprises a heavy chain-only antibody, such as a single domain antibody comprising a VH domain or a VHH domain.

[0224] In some embodiments, unless otherwise indicated, the amino acids are assigned to each domain, framework region, and CDR according to one of the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Edition), 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 Edition, Wily-VCH Verlag GmbH and Co or AbM (Oxford Molecular / MSI Pharmacopia). This does not mean 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 comprises a single domain antibody, such as a heavy chain-only antibody, such as a VH or VHH domain, and comprises three CDRs. In some embodiments, such heavy chain-only antibodies bind DLL3 as a monomer, and their optimal binding affinity is independent of dimerization with a VL (light chain variable) region.

[0225] "Variable domain residue numbering according to Kabat" or "amino acid position numbering according to Kabat" and variations thereof refer to the numbering system for heavy chain variable domains or light chain variable domains compiled in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition 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, which correspond to shortening of the FR or CDR of the variable domain or insertion therein. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of the residues of a given antibody can be determined by aligning the antibody sequence with a "standard" Kabat numbered sequence in a region of homology.

[0226] The term "framework" or "FR" residues (or regions) refers to variable domain residues other than CDR or hypervariable region residues as defined herein. A "human consensus framework" is a framework representing the most commonly occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences.

[0227] As used herein, the term " amino acid sequence identity percentage (%) " for sequence is defined as the percentage that the amino acid residue in the candidate sequence is identical with the amino acid residue in the specific sequence after the sequence is compared and introduced into room (if necessary) to reach maximum sequence identity percentage, and any conservative substitution is not considered to be a part for sequence identity. The comparison for determining the amino acid sequence identity percentage purpose can be realized by the various ways within the technical scope of the art, for example, using publicly available computer software, such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required for maximum comparison on the total length of the compared sequence.

[0228] As used herein, "elimination half-life" is used in its ordinary sense, as described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman and Alfred Gilman, eds., 6th edition 1980). In short, the term is intended to include a quantitative measure of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first-order kinetics) because the drug concentration is usually not close to the concentration required for saturation of the elimination process. The rate of an exponential process can be represented by its rate constant k or by its half-life t1 / 2, where the rate constant k represents the fractional change per unit time, and the half-life t1 / 2 represents the time required for the process to complete 50%. The units of these two constants are time-1 and time, respectively. The first-order rate constant and half-life of the reaction are simply related (k×t1 / 2=0.693) and can be interchanged accordingly. Since first-order elimination kinetics indicate that a constant fraction of drug is lost per unit time, a plot of the logarithm of drug concentration versus time is linear 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 plot.

[0229] As used herein, the term "binding affinity" refers to the affinity of a protein described in the present disclosure for its binding target, and is expressed numerically using a "Kd" value. If two or more proteins are shown to have comparable binding affinities for their binding targets, then the Kd values ​​for binding of each protein to its binding target are within ±2 times of each other. If two or more proteins are shown to have comparable binding affinities for a single binding target, then the Kd values ​​for binding of each protein to the single binding target are within ±2 times of each other. If a protein is shown to bind two or more targets with comparable binding affinities, then the Kd values ​​for binding of the protein to the two or more targets are within ±2 times of each other. Generally, higher Kd values ​​correspond to weaker binding. In some embodiments, using BIAcore TM -2000 or BIAcore TM "Kd" is measured by radiolabeled antigen binding assay (RIA) or surface plasmon resonance assay using a BIAcore-3000 (BIAcore, Inc., Piscataway, NJ). In certain embodiments, BIAcore TM -2000 or BIAcore TMThe "association rate" or "association rate" or "association rate" or "kon" and the "dissociation rate" or "dissociation rate" or "dissociation rate" or "koff" are measured by surface plasmon resonance technology using a BIAcore-3000 (BIAcore, Inc., Piscataway, NJ). In another embodiment, the Systems (Pall Life Sciences) to determine "Kd", "kon", and "koff". In an exemplary method for measuring binding affinity using a PCR system, a ligand (e.g., biotinylated human or cynomolgus monkey DLL3) is immobilized on The streptavidin capillary sensor tip is then activated using about 20-50 μg / ml of human or cynomolgus monkey DLL3 protein according to the manufacturer's instructions. A PBS / casein solution is also introduced as a blocking agent. For association kinetics measurements, DLL3 binding protein variants are introduced at a concentration 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, DLL3 binding single domain proteins are used at a concentration of about 2 ng / mL to about 20 μg / mL. Complete dissociation is observed in the case of a negative control, i.e., an assay buffer without binding protein. The kinetic parameters of the binding reaction are then determined using an appropriate tool, such as ForteBio software.

[0230] One embodiment provides a DLL3 binding protein (also referred to herein as a DLL3 binding domain, such as the DLL3 binding domain of a DLL3 trispecific antibody of the present disclosure) comprising a single domain antibody comprising a CDR1 sequence comprising a sequence selected from the group consisting of SEQ ID Nos. 443-884 and 1887, a CDR2 sequence comprising a sequence selected from the group consisting of SEQ ID Nos. 885-1326 and 1888, and a CDR3 sequence comprising a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889. In some embodiments, it is contemplated that the DLL3 binding proteins of the present disclosure are quite small, and in some embodiments are no greater than 25 kDa, no greater than 20 kDa, no greater than 15 kDa, or no greater than 10 kDa. In some cases, if it is a peptide or small molecule entity, the EGFR binding is 5 kDa or less.

[0231] In one aspect, the DLL3 targeting trispecific protein (also referred to herein as a DLL3 binding trispecific protein, a DLL3 trispecific protein, or a DLL3 TriTAC) is TM) 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. Therefore, considering that the domain order of the DLL3 targeting trispecific protein is:

[0232] H2N-(A)-(B)-(C)-COOH,

[0233] H2N-(A)-(C)-(B)-COOH,

[0234] H2N-(B)-(A)-(C)-COOH,

[0235] H2N-(B)-(C)-(A)-COOH,

[0236] H2N-(C)-(B)-(A)-COOH or

[0237] H2N-(C)-(A)-(B)-COOH.

[0238] In some embodiments, the DLL3 targeting tri-specific protein has a domain order of H2N-(A)-(B)-(C)-COOH. In some embodiments, the DLL3 targeting tri-specific protein has a domain order of H2N-(A)-(C)-(B)-COOH. In some embodiments, the DLL3 targeting tri-specific protein has a domain order of H2N-(B)-(A)-(C)-COOH. In some embodiments, the DLL3 targeting tri-specific protein has a domain order of H2N-(B)-(C)-(A)-COOH. In some embodiments, the DLL3 targeting tri-specific protein has a domain order of H2N-(C)-(B)-(A)-COOH. In some embodiments, the DLL3 targeting tri-specific protein has a domain order of H2N-(C)-(A)-(B)-COOH. In some embodiments, the DLL3 targeting tri-specific protein has a domain order of H2N-(C)-(A)-(B)-COOH.

[0239] In some embodiments, the DLL3 targeting trispecific protein has an HSA (also referred to herein as ALB) binding domain as the middle domain, such that the domain order is H2N-(A)-(B)-(C)-COOH or H2N-(C)-(B)-(A)-COOH. It is expected that in such embodiments where the HSA binding domain is the middle domain, the CD3 and DLL3 binding domains are endowed with additional flexibility to bind to their respective targets.

[0240] In some embodiments, the trispecific binding protein comprises a third domain that specifically binds DLL3, which in some cases is a single domain antibody that binds to DLL3, and which binds to DLL3 with the same or better affinity than a reference DLL3 binding parent molecule. In some embodiments, the third domain comprises an affinity matured DLL3 binding molecule (e.g., an affinity matured single domain antibody that binds to DLL3) and is derived from a DLL3 binding parent molecule that comprises one or more amino acid mutations (e.g., stabilizing mutations, destabilizing mutations) relative to the DLL3 binding parent molecule. In some embodiments, the affinity matured DLL3 binding molecule has better stability relative to a selected destabilizer than the reference DLL3 binding parent molecule. In some embodiments, the affinity matured DLL3 binding molecule is identified in a process that comprises panning one or more pre-candidate DLL3 binding molecules derived from one or more DLL3 binding parent molecules expressed in a phage display library against a DLL3 protein, such as a human DLL3 protein. In some embodiments, the precandidate DLL3 binding molecule comprises amino acid substitutions in the variable region, CDR, or framework residues relative to the parent molecule.

[0241] As used herein, "phage display" refers to a technique by which variant polypeptides are displayed as fusion proteins to at least a portion of a coat protein on the surface of a phage, filamentous phage, or particle. The utility of phage display lies in the fact that large libraries of randomized protein variants can be quickly and efficiently selected for sequences that bind to a target molecule with high affinity. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides for polypeptides with specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins by fusion with gene III or gene VIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct. Biol, 3: 355-362 (1992), and references cited therein. In monovalent phage display, a protein or peptide library is fused to gene III or a portion thereof and expressed at low levels in the presence of wild-type gene III protein, so that the phage particle displays a copy of the fusion protein or does not display the fusion protein. Relative to multivalent phage, avidity effects are reduced, so selection is based on intrinsic ligand affinity, and phagemid vectors are used, simplifying DNA manipulations. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).

[0242] In some embodiments, panning comprises 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 comprises using varying wash times to identify DLL3 binding molecules with increased or decreased off-rates from pre-candidate DLL3 molecules. In some embodiments, panning comprises using both varying binding times and varying wash times. In some embodiments, one or more stabilizing mutations are combined to increase the stability of the affinity matured DLL3 binding molecules, for example by shuffling to create a second stage combinatorial library from such mutants and performing a second round of panning followed by binding selection.

[0243] In some embodiments, the affinity matured DLL3 binding molecule has the same or better affinity to a DLL3 protein (such as a human DLL3 protein) than a DLL3 binding parent molecule, but has reduced cross-reactivity, or in some embodiments, increased cross-reactivity, with a selected substance, such as a ligand, protein, antigen, etc., that is not a DLL3 epitope for which the DLL3 binding parent molecule is specific or designed to be specific. With respect to the latter, in some embodiments, the affinity matured DLL3 binding molecule is more successfully tested in an animal model if it reacts with human DLL3 and the corresponding target of the animal model, mouse DLL3 or cynomolgus monkey DLL3. In some embodiments, the parent DLL3 binding molecule binds to human DLL3 with an affinity of about 10 nM or less and binds to cynomolgus monkey DLL3 with an affinity of about 15 nM or less. In some embodiments, the affinity matured DLL3 binding molecules identified after one round of panning bind to human DLL3 with an affinity of about 5 nM or less and bind to cynomolgus monkey DLL3 with an affinity of about 7.5 nM or less. In some embodiments, the affinity matured DLL3 binding molecules identified after two rounds of panning bind to human DLL3 with an affinity of about 2.5 nM or less and bind to cynomolgus monkey DLL3 with an affinity of about 3.5 nM or less.

[0244] In some embodiments, domain A, domain B, and domain C of the trispecific binding proteins of the present disclosure are independently antigen-specific binding domains that specifically bind to targets, such as targets on diseased cells or targets on other cells that support a disease state, such as targets on stromal cells that support tumor growth or targets on immune cells that support disease-mediated immunosuppression. In some examples, the antigen-specific binding domains include antibodies, heavy chain-only antibodies, including single-chain antibodies, Fabs, Fvs, T cell receptor binding domains, ligand binding domains, receptor binding domains, domain antibodies, single domain antibodies, minibodies, nanobodies, peptibodies, or various other antibody mimetics (such as affimers, affitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, Kunitz domain-based proteins, avimers, knottins, fynomers, darpins, affibodies, affilins, monobodies, and armadillo repeat protein-based proteins).

[0245] 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, subsequences thereof, and variants 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 higher homology with a sequence selected from SEQ ID No. 1-442 and 1886, subsequences thereof, and variants 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 higher homology with a sequence selected from SEQ ID No. 1-442 and 1886, subsequences thereof, and variants 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 higher identity to a sequence selected from SEQ ID No. 1-442 and 1886, subsequences thereof, and variants 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 higher identity to a sequence selected from SEQ ID No. 1-442 and 1886, subsequences thereof, and variants thereof.

[0246] The DLL3 targeting trispecific proteins described herein are designed to allow specific targeting of cells expressing DLL3 by recruiting cytotoxic T cells. In some embodiments, this improves efficacy compared to ADCC (antibody-dependent cell-mediated cytotoxicity), which utilizes full-length antibodies against a unique antigen and cannot directly recruit cytotoxic T cells. Instead, by engaging CD3 molecules specifically expressed on these cells, the DLL3 targeting trispecific proteins can cross-link cytotoxic T cells with cells expressing DLL3 in a highly specific manner, thereby directing the cytotoxic potential of T cells to target cells. The DLL3 targeting trispecific proteins described herein engage cytotoxic T cells via binding to surface-expressed CD3 proteins, which form part of the TCR. Simultaneous binding of several DLL3 trispecific antigen-binding proteins to CD3 and DLL3 expressed on the surface of specific cells causes T cell activation and mediates subsequent lysis of specific DLL3-expressing cells. Therefore, it is expected that DLL3 targeting trispecific proteins show strong, specific and effective target cell killing. In some embodiments, the DLL3 targeting trispecific proteins described herein stimulate cytotoxic T cells to kill target cells to eliminate pathogenic cells (e.g., tumor cells expressing DLL3). In some such embodiments, cells are selectively eliminated, thereby reducing the potential for toxic side effects.

[0247] Compared to traditional monoclonal antibodies and other smaller bispecific molecules, the DLL3-targeted trispecific proteins described herein provide further therapeutic advantages. In general, the effectiveness of recombinant protein drugs depends largely on the intrinsic pharmacokinetics of the protein itself. One such advantage is that the DLL3-targeted trispecific proteins described herein have an extended pharmacokinetic elimination half-life due to having a half-life extension domain, such as a domain that specifically binds to serum albumin (e.g., human serum albumin, HSA). In this regard, in some embodiments, the DLL3-targeted trispecific proteins described herein have an extended serum elimination half-life of about two days, three days, about five days, about seven days, about 10 days, about 12 days, or about 14 days. This is in contrast to other binding proteins such as BiTE or DART molecules that have a relatively much shorter elimination half-life. For example, the BiTE CD19×CD3 bispecific scFv-scFv fusion molecule requires continuous intravenous infusion (iv) drug delivery due to its short elimination half-life. The longer intrinsic half-life of the DLL3-targeting trispecific protein addresses this problem, allowing for increased therapeutic potential such as lower-dose drug formulations, reduced regular dosing, and / or novel pharmaceutical compositions.

[0248] The DLL3 targeting tri-specific proteins described herein also have an optimal size for enhanced tissue penetration and tissue distribution. Larger sizes limit or prevent the penetration or distribution of proteins in target tissues. The DLL3 targeting tri-specific proteins described herein avoid this problem by having a small size that allows enhanced tissue penetration and distribution. Therefore, in some embodiments, the size of the DLL3 targeting tri-specific proteins described herein is about 50kDa to about 80kDa, about 50kDa to about 75kDa, about 50kDa to about 70kDa, or about 50kDa to about 65kDa. In some embodiments, the size of the DLL3 targeting tri-specific protein is less than about 60kDa. Therefore, the size of the DLL3 targeting tri-specific protein is superior to the IgG antibody of about 150kDa and the BiTE and DART diabody molecules of about 55kDa but without an extended half-life and thus rapid clearance through the kidneys.

[0249] In further embodiments, the DLL3 targeting tri-specific proteins described herein have an optimal size for enhanced tissue penetration and distribution. In these embodiments, the DLL3 targeting tri-specific proteins are constructed to be as small as possible while retaining specificity for their target. Thus, in these embodiments, the DLL3 targeting tri-specific proteins described herein have a size of about 20 kDa to about 40 kDa, or about 25 kDa to about 35 kDa to about 40 kDa, to about 45 kDa, to about 50 kDa, to about 55 kDa, to about 60 kDa, to about 65 kDa. In some embodiments, the size of the DLL3 targeting trispecific proteins described herein is 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. An exemplary method to achieve a small size is to use a single domain antibody (sdAb) fragment for each domain. For example, a particular DLL3 trispecific antigen binding protein has an anti-CD3 sdAb, an anti-ALB sdAb, and an sdAb against DLL3. This reduces the size of the exemplary DLL3 trispecific antigen binding protein to less than 40 kDa. Thus, in some embodiments, the domains of the DLL3 targeting trispecific protein are all single domain antibody (sdAb) fragments. In some embodiments, it is contemplated that the DLL3 binding protein is quite small, and in some embodiments is no greater than 25 kDa, no greater than 20 kDa, no greater than 15 kDa, or no greater than 10 kDa. In some cases, the DLL3 binding protein, if a peptide or small molecule entity, is 5 kDa or less.

[0250] In other embodiments, the DLL3 targeting trispecific proteins described herein comprise small molecule entity (SME) conjugates for ALB, DLL3, CD3, or all. SME conjugates are small molecules with an average size of about 500Da to 2000Da, and are attached to the DLL3 targeting trispecific proteins by known methods such as sortase ligation or conjugation. In these examples, one of the domains of the DLL3 trispecific antigen binding protein is a sortase recognition sequence, LPETG (SEQ ID NO.1896). To attach the SME conjugate to the DLL3 trispecific antigen binding protein having a sortase recognition sequence, the protein is incubated with a sortase and the SME conjugate, whereby the sortase attaches the SME conjugate to the recognition sequence. In yet other embodiments, the DLL3 binding domain of the DLL3 targeting trispecific proteins described herein comprises a knottin peptide for binding to DLL3. Knottin is a disulfide-stabilized peptide with a cysteine ​​knot scaffold and has an average size of about 3.5kDa. Knottin has been predicted to bind to certain tumor molecules such as DLL3.In a further embodiment, the DLL3-binding domain of the DLL3-targeting trispecific protein described herein comprises a natural DLL3 ligand.

[0251] Another feature of the DLL3 targeting trispecific proteins described herein is that they are designed as a single polypeptide with flexible connections of their domains. This allows for easy generation and preparation of DLL3 targeting trispecific proteins, as they can be encoded by a single cDNA molecule that is easily introduced into a vector. Additionally, since the DLL3 targeting trispecific proteins described herein are monomeric single polypeptide chains, there are no chain pairing issues or dimerization required. The DLL3 targeting trispecific proteins described herein are expected to have a reduced tendency to aggregate, unlike other reported molecules, such as bispecific proteins with Fc-gamma immunoglobulin domains.

[0252] In the DLL3 targeting trispecific proteins described herein, the domains are connected by internal linkers L1 and L2 in some embodiments, wherein L1 connects the first and second domains of the DLL3 targeting trispecific protein, and L2 connects the second and third domains of the DLL3 targeting trispecific protein. Linkers L1 and L2 have optimized lengths and / or amino acid compositions. 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, internal linkers L1 and / or L2 are "short", i.e., consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, 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, internal linkers L1 and / or L2 are "long", i.e., consisting of 15, 20 or 25 amino acid residues. In some embodiments, these internal linkers consist of about 3 to about 15, such as 8, 9 or 10 consecutive amino acid residues. Regarding the amino acid composition of the internal linkers L1 and L2, peptides are selected that have the following properties, namely, they can confer flexibility to the DLL3 targeting trispecific protein, do not interfere with the binding domain, and resist protease cleavage. For example, glycine and serine residues generally provide protease resistance. Examples of internal linkers suitable for connecting domains in DLL3 targeting trispecific proteins include, but are not limited to (GS) n (SEQ ID No. 1809), (GGS) n (SEQ ID No.1810), (GGGS) n (SEQ ID No.1811), (GGSG) n (SEQ ID No.1812), (GGSGG) n (SEQ ID No.1813), (GGGGS) n )SEQ ID No.1814)、(GGGGG) n (SEQ ID No. 1815) or (GGG) n (SEQ ID No.1816), wherein 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).

[0253] In some cases, enzymatic site-specific conjugation methods are used to conjugate domains within the DLL3 targeting trispecific protein, which involve the use of mammalian or bacterial transglutaminases. Microbial transglutaminases (mTG) are versatile tools in modern research and biotechnology. The availability of large amounts of relatively pure enzymes, ease of use, and lack of regulation by calcium and guanine-5'-triphosphate (GTP) have prompted mTG to become the main cross-linking enzyme used in the food industry and biotechnology. Currently, mTG is used in many applications to attach proteins and peptides to small molecules, polymers, surfaces, DNA, and other proteins. See, Pavel Strp, Veracity of microbial transglutaminase, Bioconjugate Chem. 25, 5, 855-862).

[0254] In some examples, DLL3 targeting trispecific proteins are provided, wherein one of the domains contains an acceptor glutamine in the constant region, which can then be conjugated to the other domain via a lysine-based linker (e.g., any primary amine chain as a TGase substrate, including alkylamines, oxoamines), wherein conjugation occurs only on one or more acceptor glutamine residues present in the targeting portion outside the antigen binding site (e.g., outside the variable region, in the constant region). Thus, conjugation does not occur on glutamines (at least partially surface-exposed glutamines) within the variable region. In some examples, a trispecific protein is formed by reacting one of the domains with a lysine-based linker in the presence of TGase.

[0255] In some embodiments, where one or more domains within the DLL3 targeting trispecific binding protein are directly joined, a hybrid vector is prepared in which the DNA encoding the directly joined domains themselves are directly linked to each other. In some embodiments, where a linker is used, a hybrid vector is prepared in which the DNA encoding the first of the three domains is linked to the DNA encoding one end of the first linker portion, and the DNA encoding the second of the three domains is linked to the other end of the first linker portion; further, the DNA encoding the second of the three domains is linked to one end of the second linker portion, and the DNA encoding the third of the three domains is linked to the other end of the second linker portion, wherein the first domain, the second domain, and the third domain are different, and wherein the first domain, the second domain, and the third domain are independently selected from domain A, domain B, and domain C. Such a connection is performed, for example, in series or as a three-way connection.

[0256] CD3 binding domain

[0257] The specificity of the T cell response is mediated by the recognition of antigens (displayed in the context of the major histocompatibility complex MHC) by the TCR. As part of the TCR, CD3 is a protein complex containing the CD3γ(gamma) chain, the CD3δ(delta) chain, and two CD3ε(epsilon) chains present on the cell surface. CD3 associates with the α(alpha) and β(beta) chains of the TCR and together with CD3ζ(zeta) constitutes the complete TCR. Clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodies, results in T cell activation, which is similar to the engagement of the T cell receptor, but is independent of its clone-specific specificity.

[0258] In one aspect, the DLL3 targeting tri-specific proteins described herein comprise a domain that specifically binds to CD3. In one aspect, the DLL3 targeting tri-specific proteins described herein comprise a domain that specifically binds to human CD3. In some embodiments, the DLL3 targeting tri-specific proteins described herein comprise a domain that specifically binds to CD3γ. In some embodiments, the DLL3 targeting tri-specific proteins described herein comprise a domain that specifically binds to CD3δ. In some embodiments, the DLL3 targeting tri-specific proteins described herein comprise a domain that specifically binds to CD3ε.

[0259] In further embodiments, the DLL3 targeting trispecific proteins described herein comprise a domain that specifically binds to a TCR. In certain instances, the DLL3 targeting trispecific proteins described herein comprise a domain that specifically binds to the α chain of a TCR. In certain instances, the DLL3 targeting trispecific proteins described herein comprise a domain that specifically binds to the β chain of a TCR.

[0260] In certain embodiments, the CD3 binding domain of the DLL3 targeting trispecific proteins described herein not only exhibits potent CD3 binding affinity for human CD3, but also shows excellent cross-reactivity with the respective cynomolgus CD3 proteins.

[0261] 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 cases, it is beneficial that the CD3 binding domain is derived from the same species in which the DLL3 trispecific antigen binding protein will ultimately be used. For example, for human use, it may be beneficial for the CD3 binding domain of the DLL3 trispecific antigen binding protein to comprise human or humanized residues from the antigen binding domain of an antibody or antibody fragment.

[0262] Thus, in one aspect, the antigen binding domain comprises a humanized or human antibody or antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-CD3 binding domain comprises one or more (e.g., all three) of the light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 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 complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of the humanized or human anti-CD3 binding domain described herein, the humanized or human anti-CD3 binding domain comprises one or more, all three LC CDRs, and one or more, all three HC CDRs.

[0263] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human light chain variable region specific for CD3, wherein the light chain variable region specific for CD3 comprises a human or non-human light chain CDR in a human light chain framework region. In certain instances, the light chain framework region is a lambda light chain framework. In other instances, the light chain framework region is a kappa light chain framework.

[0264] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human heavy chain variable region that is specific for CD3, wherein the heavy chain variable region that is specific for CD3 comprises human or non-human heavy chain CDRs in the human heavy chain framework region.

[0265] In certain examples, the complementarity determining regions of the heavy and / or light chains are derived from known anti-CD3 antibodies, e.g., muromonab CD3 (OKT3), otelizumab (TRX4), telizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, and WT-31.

[0266] In one embodiment, the anti-CD3 binding domain is a single chain variable fragment (scFv) of a light chain and a heavy chain comprising an amino acid sequence provided herein. As used herein, a "single chain variable fragment" or "scFv" refers to an antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain and heavy chain variable regions are continuously connected via a short flexible polypeptide connector and can be expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. In one embodiment, the anti-CD3 binding domain comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95%-99% identity to an amino acid sequence provided herein; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95%-99% identity to an amino acid sequence provided herein. In some examples, the anti-CD3 binding domain comprises a sequence selected from SEQ ID 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 with 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 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 attached to the heavy chain variable region comprising the amino acid sequence described herein via an scFv connector. The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: light chain variable region-scFv connector-heavy chain variable region or heavy chain variable region-scFv connector-light chain variable region.

[0267] In some examples, scFvs that bind to CD3 are prepared according to known methods. For example, scFv molecules can be produced by connecting VH and VL regions together using a flexible polypeptide connector. The scFv molecule comprises an scFv connector (e.g., a Ser-Gly connector) having an optimized length and / or amino acid composition. Therefore, in some embodiments, the length of the scFv connector allows the VH or VL domain to be intermolecularly associated with other variable domains to form a CD3 binding site. In certain embodiments, such scFv connectors are "short", i.e., composed of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Therefore, in certain examples, the scFv connector is composed of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the scFv connector is a peptide bond. In some embodiments, these scFv connectors are composed of about 3 to about 15, for example 8, 9 or 10 continuous amino acid residues. Regarding the amino acid composition of the scFv linker, peptides are selected that confer flexibility, do not interfere with the variable domains, and allow interchain folding so that the two variable domains together form a functional CD3 binding site. For example, scFv linkers comprising glycine and serine residues generally provide protease resistance. In some embodiments, the linker in the scFv comprises glycine and serine residues. The amino acid sequence of the scFv linker can be optimized by, for example, phage display methods to improve CD3 binding and yield of the scFv. Examples of peptide scFv linkers suitable for connecting the variable light chain domain and the variable heavy chain domain in the 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), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the scFv linker can be (GGGGS) 4 (SEQ ID No. 1817) or (GGGGS) 3 (SEQ ID No. 1818). In some embodiments, the linker comprises a sequence consisting of any combination of linkers shown in SEQ ID No. 1809-1818, and in some examples, the length of such a linker is up to 15 amino acids, or longer than 15 amino acids. Variations in linker length can maintain or enhance activity, thereby producing superior efficacy in activity studies.

[0268] In some embodiments, the CD3 binding domain of the DLL3 targeting trispecific antigen binding protein has an affinity for CD3 on cells expressing CD3 with a K D 5 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, 0.5 nM or less, or 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 targeting trispecific antigen binding protein has an affinity for CD3ε, CD3γ, or CD3δ with a K D In a further embodiment, the CD3 binding domain of the DLL3 targeting trispecific antigen binding protein has a low affinity for CD3, i.e., about 100 nM or more.

[0269] The affinity for binding to CD3 can be determined by, for example, the ability of the DLL3-targeting trispecific antigen-binding protein itself or its CD3-binding domain to bind to CD3 coated on an assay plate, CD3 displayed on the surface of a microbial cell, CD3 in a solution, etc. The binding activity of the DLL3-targeting trispecific antigen-binding protein itself or its CD3-binding domain to CD3 of the present disclosure can be determined as follows: a ligand (e.g., CD3) or the DLL3-targeting trispecific antigen-binding protein itself or its CD3-binding domain is immobilized to beads, a substrate, a cell, etc. The reagent can be added to a suitable buffer, and the binding partner is incubated at a given temperature for a period of time. After washing to remove unbound substances, the bound protein can be released using, for example, SDS, a high pH buffer, etc., and analyzed by, for example, surface plasmon resonance (SPR).

[0270] Half-life extension domain

[0271] Concerning domains that extend the half-life of antigen binding domains, it is contemplated that such domains include, but are not limited to, albumin binding domains, Fc domains, small molecules, and other half-life extension domains known in the art.

[0272] Human albumin (ALB) (molecular weight 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 serves to maintain plasma pH, contributes to colloid blood pressure, acts as a carrier for many metabolites and fatty acids, and serves as the major drug transporter in plasma.

[0273] Non-covalent association with albumin prolongs the elimination half-life of short-lived proteins. For example, recombinant fusions of albumin binding domains with Fab fragments resulted in 25-fold and 58-fold increased in vivo clearance and 26-fold and 37-fold increased half-life when administered intravenously to mice and rabbits, respectively, compared to administration of the Fab fragment alone. In another example, when insulin was acylated with fatty acids to promote association with albumin, a prolonged effect was observed when injected subcutaneously in rabbits or pigs. In summary, these studies demonstrate a link between albumin binding and a prolonged effect.

[0274] In one aspect, the DLL3 targeting trispecific proteins described herein comprise a half-life extension domain, such as a domain that specifically binds to ALB. In some embodiments, the ALB binding domain of the DLL3 targeting trispecific antigen binding protein can be any domain that binds to ALB, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. In some embodiments, the ALB binding domain is a single chain variable fragment (scFv), a single domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of a single domain antibody of camelid origin, a peptide, a ligand, or a small molecule entity that is specific for HSA. In certain embodiments, the ALB binding domain is a single domain antibody. In other embodiments, the ALB binding domain is a peptide. In further embodiments, the ALB binding domain is a small molecule. In some embodiments, it is envisioned that the HSA binding domain of the DLL3 trispecific antigen binding protein is quite small and does not exceed 25 kD, does not exceed 20 kDa, does not exceed 15 kDa, or does not exceed 10 kDa. In certain instances, the ALB binding protein, if a peptide or small molecule entity, is 5 kDa or smaller.

[0275] The half-life extension domain of the DLL3 targeting trispecific antigen binding protein results in pharmacodynamic and pharmacokinetic changes to the DLL3 targeting 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 pharmacodynamic properties, including changes in tissue distribution, penetration and diffusion of the trispecific antigen binding protein. In some embodiments, the half-life extension domain provides improved tissue (including tumor) targeting, tissue distribution, tissue penetration, diffusion within tissues, and enhanced efficacy compared to proteins 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 reduced side effects, such as reduced cytotoxicity to non-tumor cells.

[0276] In addition, the binding affinity of the half-life extension domain can be selected so as to target a specific elimination half-life in a specific trispecific antigen binding protein. Therefore, in some embodiments, the half-life extension domain has a high binding affinity. In other embodiments, the half-life extension domain has a medium binding affinity. In other embodiments, the half-life extension domain has a low or tiny binding affinity. Exemplary binding affinities include 10nM or lower (high), 10nM to 100nM (medium) and greater than 100nM (low) KD concentrations. As described above, the binding affinity to ALB is determined by known methods such as surface plasmon resonance (SPR). In some embodiments, the ALB binding domain described herein comprises a single domain antibody.

[0277] In some embodiments, the half-life extension domain comprises 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 domain comprises three heavy chain CDRs (HC CDR1, HC CDR2 and HC CDR3) and three light chain CDRs. In some examples, the half-life extension domain 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 comprises 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 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 in a sequence selected from SEQ ID No. 1791 or 1792.

[0278] DLL3 binding domain

[0279] 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, humans (accession numbers NP_058637 and NP_982353), chimpanzees (accession number XP_003316395), mice (accession number NP_031892), and rats (accession number NP_446118). In humans, the DLL3 gene consists of 8 exons located on chromosome 19q13, spanning 9.5 kbp. Alternative splicing within the last exon produces two processed transcripts, one of 2389 bases (accession number NM_016941) and the other of 2052 bases (accession number NM_203486). The former transcript encodes a protein of 618 amino acids (Accession No. NP_058637), while the latter encodes a protein of 587 amino acids (Accession No. NP_982353). The two protein isoforms of DLL3 share 100% identity in their extracellular and transmembrane domains, differing only in that the longer isoform contains an extended cytoplasmic tail that contains an additional 32 residues at the carboxyl terminus of the protein. The extracellular region of the DLL3 protein contains six EGF-like domains, a single DSL domain, and an N-terminal domain. Generally, EGF domains are identified as occurring at approximately amino acid residues 216-249 (domain 1), 274-310 (domain 2), 312-351 (domain 3), 353-389 (domain 4), 391-427 (domain 5), and 429-465 (domain 6), with the DSL domain being located at approximately amino acid residues 176-215 of hDLL3 and the N-terminal domain being located at approximately amino acid residues 27-175 of hDLL3. Each of the EGF-like domains, the DSL domain, and the N-terminal domain comprise a portion of the DLL3 protein defined by a different amino acid sequence. In some embodiments, the EGF-like domains are referred to as EGF1 to EGF6, with EGF1 being closest to the N-terminal portion of the protein. In general, DSL ligands consist of a series of domains: a unique N-terminal domain followed by a conserved DSL domain, multiple tandem epidermal growth factor (EGF)-like repeats, a transmembrane domain, and a cytoplasmic domain that is not highly conserved between ligands, but one of which contains multiple lysine residues that are potential sites of ubiquitination by a unique E3 ubiquitin ligase. The DSL domain is a degenerate EGF domain that is necessary but not sufficient for interaction with the Notch receptor. In addition, the first two EGF-like repeats of most DSL ligands contain a smaller protein sequence motif, called the DOS domain, which acts synergistically with the DSL domain in activating Notch signaling.

[0280] In some embodiments, the DLL3 trispecific binding proteins disclosed herein are generated, prepared, engineered or selected to react with a selected domain, motif or epitope within a DLL3 protein. In some embodiments, the DLL3 targeting trispecific protein binds to a DSL domain, and in some embodiments, to an epitope within the DSL domain comprising G203, R205, P206.

[0281] In some embodiments, the binding domains of the DLL3 targeting trispecific proteins of the present disclosure are engineered and / or selected to react with both isoforms of DLL3 or a single isoform of the protein, or conversely, comprise a pan-DLL binding domain that reacts or associates with at least one additional DLL family member in addition to DLL3. In some embodiments, DLL3 binding domains, such as DLL3 binding domains, are engineered, prepared and / or selected such that they react with domains (or epitopes therein) that are displayed only by DLL3, or with domains that are conserved, at least to some extent, among multiple or all DLL family members.

[0282] In some embodiments, the DLL3 binding domain associates or binds to a specific epitope, portion, motif, or domain of DLL3. Both DLL3 isoforms comprise the same extracellular region, which comprises at least an N-terminal domain, a DSL (Delta / Serrate / lag-2) domain, and six EGF-like domains (i.e., EGF1-EGF6). Thus, in certain embodiments, the DLL3 binding domain associates or binds to the N-terminal domain of DLL3 (amino acids 27-175 in the mature protein), while in other embodiments, the DLL3 binding domain associates with the DSL domain (amino acids 176-215) or an epitope therein. In other aspects of the disclosure, the DLL3 binding domain associates or binds to a specific epitope located in a specific EGF-like domain of DLL3. In some embodiments, the DLL3 binding domain associates or binds to an epitope located in EGF1 (amino acids 216-249), EGF2 (amino acids 274-310), EGF3 (amino acids 312-351), EGF4 (amino acids 353-389), EGF5 (amino acids 391-427), or EGF6 (amino acids 429-465). In some embodiments, each of the above domains comprises more than one epitope and / or more than one bin. In some embodiments, the DLL3 binding domain binds, reacts, or associates with a DSL domain or an epitope therein. In other embodiments, the DLL3 binding domain binds, reacts, or associates with a specific EGF-like domain or an epitope therein. In some embodiments, the DLL3 binding domain binds, reacts, or associates with an N-terminal domain or an epitope therein.

[0283] In some embodiments, a DLL3 binding protein of the present disclosure, such as a DLL3 binding domain of a trispecific protein of the present disclosure, binds to a full-length DLL3 protein or a fragment thereof, such as a fragment comprising an epitope within a full-length DLL3 protein, as described above. In some cases, the fragment comprising an epitope comprises an antigenic or immunogenic fragment of a DLL3 protein and derivatives thereof. In some embodiments, the fragment comprising an antigenic determinant, including an antigenic or immunogenic fragment, is 12 amino acids or more, 20 amino acids or more, 50 or 100 amino acids or more. In some embodiments, the DLL3 fragment is 95% or more of the length of the complete protein, 90% or more of the length of the complete protein, 75% or 50% or 25% or 10% or more. In some embodiments, the fragment comprising an epitope of DLL3, including an antigenic or immunogenic fragment, is capable of eliciting a relevant immune response in a patient. In some embodiments, derivatives of DLL3 include sequence variants in which one or more (e.g., 1-20, such as 15 amino acids, or up to 20%, such as up to 10%, 5%, or 1%, based on the total length of the protein in terms of the number of amino acids) deletions, insertions, or substitutions have been made to the DLL3 sequence provided in SEQ ID No. 1885 (UniProtKB Accession Q9NYJ7). In some embodiments, the substitutions include conservative substitutions. In some examples, derivatives and variants of DLL3 have substantially the same biological function as the DLL3 protein from which they are derived. For example, in some cases, derivatives and variants of DLL3 have comparable antigenicity or immunogenicity to the protein from which they are derived, have the ligand binding activity or active receptor complex forming ability of the protein from which they are derived, or preferably both, and have the same tissue distribution as DLL3.

[0284] The design of the DLL3 targeting trispecific proteins described herein allows for the DLL3 binding domain to be flexible, as the DLL3 binding domain can be any type of binding domain, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. In some embodiments, the DLL3 binding domain 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 of camelid origin (VHH). In other embodiments, the DLL3 binding domain is a non-Ig binding domain, i.e., an antibody mimetic, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, and monobodies. In further embodiments, the DLL3 binding domain is a ligand or peptide that binds or associates with DLL3. In further embodiments, the DLL3 binding domain is a knottin. In further embodiments, the DLL3 binding domain is a small molecule entity.

[0285] 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 sequence that is truncated compared to SEQ ID No. 1885 (UniProtKB accession Q9NYJ7). In some embodiments, the DLL3 binding domain binds to a protein comprising the sequence of SEQ ID No. 1892 or SEQ ID No. 1893, which is the mature extracellular domain of a DLL3 protein. In some embodiments, the DLL3 binding domain binds to a protein comprising amino acids 47-492 of SEQ ID No. 1892. In some embodiments, the DLL3 binding domain recognizes an epitope within amino acids 47-4492 of SEQ ID No. 1892.

[0286] 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 expected to improve the binding affinity and / or other biological properties of the antibodies. Exemplary methods for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody.

[0287] Any combination of deletion, insertion and substitution can be performed to obtain the final construct, provided that the final construct has the desired characteristics, antigen binding. In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The sites of interest for substitution mutagenesis include CDR and framework regions. Examples of such substitutions are described below. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, retained / improved antigen binding, reduced immunogenicity or improved T cell-mediated cytotoxicity (TDCC). Conservative and non-conservative amino acid substitutions are considered for the preparation of antibody variants.

[0288] In another example of substitutions used to generate variant anti-DLL3 antibodies, one or more hypervariable region residues of a parent antibody are substituted. Typically, variants are then selected based on improvement of a desired property compared to the parent antibody, e.g., increased affinity, decreased affinity, decreased immunogenicity, increased pH dependence of binding.

[0289] In some embodiments, the DLL3 binding domain of the DLL3 targeting trispecific protein is a single domain antibody, such as a heavy chain variable domain (VH) specific for DLL3, a variable domain (VHH) of a llama-derived sdAb, a peptide, a ligand, or a small molecule entity. In some embodiments, the DLL3 binding domain of the 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.

[0290] Generally, it should be noted that the term single domain antibody, as used herein in its broadest sense, is not limited to a specific biological source or a specific method of preparation. A single domain antibody is an antibody whose complementary determining region is a part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that are naturally free of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds different from those derived from antibodies. A single domain antibody can be any single domain antibody in the art, or any future single domain antibody. Single domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, cattle. For example, in some embodiments, the single domain antibodies of the present disclosure are obtained by the following methods: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, in particular from a mammalian species, such as from humans, or by expressing a nucleic acid encoding such a camelized VH domain; (5) by "camelization" of a "domain antibody" or "Dab", or by expressing a nucleic acid encoding such a camelized VH domain; (6) by preparing a protein, polypeptide or other amino acid sequence using synthetic or semi-synthetic techniques; (7) by preparing a nucleic acid encoding a single domain antibody using nucleic acid synthesis techniques known in the art, and then expressing the nucleic acid obtained thereby; and / or (8) by any combination of one or more of the foregoing.

[0291] In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody against DLL3. As further described herein, such VHH sequences can generally be generated or obtained by appropriately immunizing a llama species with DLL3 (i.e., to generate an immune response and / or heavy chain antibodies against DLL3), obtaining a suitable biological sample (such as a blood sample, serum sample or B cell sample) from the llama, and generating a VHH sequence against DLL3 starting from the sample using any suitable technique known in the art.

[0292] In another embodiment, such naturally occurring VHH domains against DLL3 are obtained from a naive library of Camelidae VHH sequences, for example by screening such a library using DLL3 or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, such as VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, for example as described in WO 00 / 43507.

[0293] In a further embodiment, yet another technique for obtaining VHH sequences against DLL3 involves appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., to generate an immune response and / or heavy chain antibodies against DLL3), obtaining a suitable biological sample (such as a blood sample, a serum sample, or a B cell sample) from the transgenic mammal, and then generating VHH sequences against DLL3 starting from the sample using any suitable technique known in the art. For example, rats or mice expressing heavy chain antibodies and other methods and techniques described in WO 02 / 085945 and WO 04 / 049794 may be used for this purpose.

[0294] In some embodiments, the DLL3 targeting trispecific protein anti-DLL3 single domain antibodies include single domain antibodies having an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VHH domain, but which has been "humanized", i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (particularly in the framework sequence) with one or more amino acid residues present at the corresponding positions in the VH domain of a conventional 4-chain antibody from a human (e.g., as described above). This can be done in a manner known in the art, which will be apparent to a person skilled in the art, e.g., based on the further description herein. In addition, it should be noted that such humanized anti-DLL3 single domain antibodies of the present disclosure are obtained in any suitable manner known per se (i.e., as indicated in points (1)-(8) above), and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VHH domains as starting materials. In some additional embodiments, the single domain anti-DLL3 antibodies as described herein include single domain antibodies having an amino acid sequence corresponding to the amino acid sequence of a naturally occurring VH domain, but which has been "camelized", i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody with one or more amino acid residues present at the corresponding positions in the VHH domain of a heavy chain antibody. Such "camelizing" substitutions are preferably inserted at amino acid positions that form the VH-VL interface and / or are present at the VH-VL interface and / or at the so-called Camelidae Hallmark residues (see, e.g., WO 94 / 04678, and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence used as the starting material or starting point for generating or designing a camelized single domain is preferably a VH sequence from a mammal, more preferably a human VH sequence, such as a VH3 sequence. However, it should be noted that, in certain embodiments, such camelized anti-DLL3 single domain antibodies of the present disclosure are obtained in any suitable manner known in the art (i.e., as indicated in points (1)-(8) above), and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VH domains as starting materials. For example, as further described herein, both "humanization" and "camelization" are performed by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then changing one or more codons in the nucleotide sequence in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" single domain antibody, respectively. The nucleic acid can then be expressed to provide the desired anti-DLL3 single domain antibody of the present disclosure. Alternatively, in other embodiments, the amino acid sequence of the desired humanized or camelized anti-DLL3 single domain antibody of the present disclosure is designed based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known peptide synthesis techniques.In some embodiments, a nucleotide sequence encoding a desired humanized or camelized anti-DLL3 single domain antibody of the present disclosure is designed based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known nucleic acid synthesis techniques, followed by expressing the nucleic acid thus obtained using known expression techniques to provide the desired anti-DLL3 single domain antibody of the present disclosure.

[0295] Other suitable methods and techniques for obtaining the anti-DLL3 single domain antibodies of the present disclosure and / or nucleic acids encoding the anti-BCMA single domain antibodies starting from naturally occurring VH sequences 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 the nucleotide sequence or nucleic acid encoding the anti-BCMA single domain antibodies.

[0296] In some embodiments, the DLL3 binding domain is an anti-DLL3 specific antibody comprising a heavy chain variable complementary determining region CDR1, a heavy chain variable CDR2, a heavy chain variable CDR3, a light chain variable CDR1, a light chain variable CDR2, and a light chain variable CDR3. In some embodiments, the DLL3 binding domain comprises any domain that binds to DLL3, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, or antigen binding fragments such as single domain antibodies (sdAb), Fab, Fab', F(ab)2, and Fv fragments, fragments consisting of one or more CDRs, single chain antibodies (e.g., single chain Fv fragments (scFv)), disulfide-stabilized Fv (dsFv) fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain. In some embodiments, the DLL3 binding domain is a single domain antibody. In some embodiments, an anti-DLL3 single domain antibody comprises a heavy chain variable complementarity determining region (CDR), CDR1, CDR2, and CDR3.

[0297] In some embodiments, the DLL3 binding domain is a polypeptide comprising an amino acid sequence consisting of four framework regions / sequences (f1-f4) interrupted by three complementary determining regions / sequences, as shown in the following formula: f1-r1-f2-r2-f3-r3-f4, wherein r1, r2 and r3 are complementary determining regions CDR1, CDR2 and CDR3, respectively, and f1, f2, f3 and f4 are framework residues. The framework residues of the DLL3 binding proteins of the present disclosure comprise, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 or 94 amino acid residues, and the complementary determining regions comprise, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 amino acid residues. In some embodiments, the DLL3 binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886. In some embodiments, the CDR1 of the DLL3 binding domain 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, the 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, the CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889, or one or more substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889.

[0298] In some embodiments, the CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 443-884 and 1887, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in an amino acid selected from the group consisting of SEQ ID Nos. 443-884 and 1887. In some embodiments, the CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 885-1326 and 1888, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in an amino acid sequence selected from the group consisting of SEQ ID Nos. 885-1326 and 1888. In some embodiments, the CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889.

[0299] In some embodiments, the CDR1 comprises an amino acid sequence selected from SEQ ID No.495-528, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in an amino acid selected from SEQ ID No.495-528. In some embodiments, the CDR2 comprises an amino acid sequence selected from SEQ ID No.937-970, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in an amino acid sequence selected from SEQ ID No.937-970. In some embodiments, the CDR3 comprises an amino acid sequence selected from SEQ ID No.1379-1412, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in a sequence selected from SEQ ID No.1379-1412.

[0300] In some embodiments, the CDR1 comprises an amino acid sequence selected from SEQ ID No.529-809, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in an amino acid selected from SEQ ID No.529-809. In some embodiments, the CDR2 comprises an amino acid sequence selected from SEQ ID No.971-1251, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in an amino acid sequence selected from SEQ ID No.971-1251. In some embodiments, the CDR3 comprises an amino acid sequence selected from SEQ ID No.1379-1412, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in a sequence selected from SEQ ID No.1379-1412.

[0301] In some embodiments, the CDR1 comprises an amino acid sequence selected from SEQ ID No.810-884, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in an amino acid selected from SEQ ID No.810-884. In some embodiments, the CDR2 comprises an amino acid sequence selected from SEQ ID No.1252-1326, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in an amino acid sequence selected from SEQ ID No.1252-1326. In some embodiments, the CDR3 comprises an amino acid sequence selected from SEQ ID No.1692-1768, or a variant having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in a sequence selected from SEQ ID No.1692-1768.

[0302] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID Nos. 1-442 and 1886. In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID No. 53-86.

[0303] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID Nos. 87-367.

[0304] 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.

[0305] 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.

[0306] In some embodiments, the DLL3 binding domain of the DLL3 targeting trispecific binding protein has cross-reactivity with human and cynomolgus DLL3. In some embodiments, the DLL3 binding domain is specific for human DLL3. In certain embodiments, the DLL3 binding domain disclosed herein binds to human DLL3 with a human Kd (hKd). In certain embodiments, the DLL3 binding domain disclosed herein binds to cynomolgus DLL3 with a cynomolgus Kd (cKd). In certain embodiments, the DLL3 binding domain disclosed herein binds to cynomolgus DLL3 and human DLL3 with a cynomolgus Kd (cKd) and a human Kd (hKd), respectively. In some embodiments, the DLL3 binding protein binds to human and cynomolgus DLL3 with comparable binding affinities (i.e., the hKd and cKd values ​​differ by no more than ±10%). In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 500 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 450 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 400 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 350 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 300 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 250 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 200 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 150 nM. In some embodiments, the hKd and cKd are in the range of about 0.001 nM to about 100 nM. In some embodiments, the hKd and cKd are in the range of about 0.1 nM to about 90 nM. In some embodiments, the hKd and cKd are in the range of about 0.2 nM to about 80 nM. In some embodiments, the hKd and cKd are in the range of about 0.3 nM to about 70 nM. In some embodiments, the hKd and cKd are in the range of about 0.4 nM to about 50 nM. In some embodiments, the hKd and cKd are in the range of about 0.5 nM to about 30 nM. In some embodiments, the hKd and cKd are in the range of about 0.6 nM to about 10 nM. In some embodiments, the hKd and cKd are in the range of about 0.7 nM to about 8 nM. In some embodiments, the hKd and cKd are in the range of about 0.8 nM to about 6 nM. In some embodiments, the hKd and cKd are in the range of about 0.9 nM to about 4 nM. In some embodiments, the hKd and cKd are in the range of about 1 nM to about 2 nM.

[0307] In certain embodiments, a DLL3 binding domain of the present disclosure preferentially binds to membrane-bound DLL3 over soluble DLL3. Membrane-bound DLL3 refers to DLL3 present in or on the cell membrane surface of a cell expressing DLL3. Soluble DLL3 refers to DLL3 that is no longer present in or on the cell membrane surface of a cell that is expressing or has expressed DLL3. In certain instances, soluble DLL3 is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the DLL3 binding protein binds to membrane-bound DLL3 at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold more than soluble DLL3. In one embodiment, an antigen binding protein of the present disclosure preferentially binds to membrane-bound DLL3 30-fold more than soluble DLL3. Preferential binding of an antigen binding protein to membrane-bound DLL3 over soluble DLL3 can be readily determined using assays known in the art.

[0308] In some embodiments, any of the aforementioned DLL3 binding domains (e.g., anti-DLL3 single domain antibodies of SEQ ID NOs. 1-442 and 1886) is an affinity peptide tagged for ease of purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his (SEQ ID No. 1819).

[0309] In some embodiments, any of the aforementioned DLL3 binding domains (e.g., anti-DLL3 single domain antibodies of SEQ ID NOs. 1-442 and 1886) is an affinity peptide tagged for ease of purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his (SEQ ID No. 1819).

[0310] Integration into chimeric antigen receptors (CARs)

[0311] In certain instances, the DLL3 targeting trispecific antigen binding protein disclosed herein can be incorporated into a chimeric antigen receptor (CAR). Engineered immune effector cells, T cells, or NK cells can be used to express a CAR, which includes an anti-DLL3 targeting trispecific protein containing an anti-DLL3 single domain antibody as described herein. In one embodiment, the CAR comprising the anti-DLL3 targeting trispecific protein described herein is connected to a transmembrane domain via a hinge region, and is further connected to a co-stimulatory domain, obtained from a functional signaling domain of OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB. In some embodiments, the CAR further comprises a sequence encoding an intracellular signaling domain such as 4-1BB and / or CD3ζ.

[0312] Tumor growth reducing properties

[0313] In certain embodiments, the DLL3-targeting trispecific proteins of the present disclosure, when administered to a subject having tumor cells expressing DLL3, reduce the growth of tumor cells in vivo. Measurement of reduction in tumor cell growth can be determined by a variety of different methods known in the art. Non-limiting examples include direct measurement of tumor size, measurement of resected tumor masses and comparison to control subjects, 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, and the like.

[0314] In a specific embodiment, compared with a control antigen binding agent, the administration of the tri-specific protein of the present disclosure results in a reduction in the in vivo growth of tumor cells of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, and a reduction in tumor growth of about 100% indicates complete response and tumor disappearance. In a further embodiment, compared with a control antigen binding agent, the administration of the tri-specific protein of the present disclosure results in a reduction in the in vivo growth of tumor cells of about 50-100%, about 75-100% or about 90-100%. In a further embodiment, compared with a control antigen binding agent, the administration of the tri-specific protein of the present disclosure results in a reduction in the in vivo growth of tumor cells of about 50-60%, about 60-70%, about 70-80%, about 80-90% or about 90-100%.

[0315] DLL3 targets trispecific protein modifications

[0316] The DLL3 targeting trispecific proteins described herein include derivatives or analogs in which (i) amino acids are replaced with amino acid residues not encoded by the genetic code, (ii) the mature polypeptide is fused to another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader sequence or a secretory sequence or a sequence for purifying the protein.

[0317] Typical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation and ubiquitination.

[0318] Modifications are made at any position in the DLL3 targeting trispecific proteins described herein, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Certain common peptide modifications that can be used to modify DLL3 targeting trispecific proteins include glycosylation, lipid attachment, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation, blocking of the amino or carboxyl groups or both in the polypeptide by covalent modification, and ADP-ribosylation.

[0319] In some embodiments, a derivative of a DLL3 targeting trispecific protein as described herein comprises an immunoresponsiveness modulator derivative and an antigen binding molecule comprising one or more modifications.

[0320] In some embodiments, the trispecific DLL3 binding molecules of the present disclosure are monovalent or multivalent, bivalent, trivalent, etc. As used herein, the term "valency" refers to the number of potential target binding sites associated with an antibody. Each target binding site specifically binds to one target molecule or a specific location or locus on a target molecule. When the antibody is monovalent, each binding site of the molecule will specifically bind to one antigen location or epitope. When the antibody comprises more than one target binding site (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 locations on the same antigen).

[0321] In some embodiments, the DLL3 targeting trispecific proteins of the present disclosure comprise, inter alia, one or more additional amino acid residue substitutions, mutations and / or modifications that result in compounds with preferred properties, including, but not limited to, altered pharmacokinetics, increased serum half-life, increased binding affinity, reduced immunogenicity, increased production, altered binding of Fc ligands to Fc receptors (FcRs), enhanced or reduced "ADCC" (antibody-dependent cell-mediated cytotoxicity) or "CDC" (complement-dependent cytotoxicity) activity, altered glycosylation and / or disulfide bonds, and altered binding specificity. In some cases, these DLL3 targeting trispecific protein variants are advantageously used to enhance the effective anti-tumor properties of the disclosed DLL3 targeting trispecific proteins.

[0322] In some embodiments, the half-life of the DLL3 targeting tri-specific protein of the present disclosure in a mammal, such as a human or a cynomolgus monkey, is 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. In some cases, the extended half-life results in higher serum titers, thereby reducing the dosing frequency of the DLL3 targeting tri-specific protein, reducing the concentration of the antibody to be administered, or both.

[0323] Still other embodiments include one or more engineered glycoforms, i.e., DLL3-targeted trispecific binding proteins comprising an altered glycosylation pattern or altered carbohydrate composition covalently linked to the protein. In some cases, engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or reducing effector function, increasing the affinity of the trispecific protein to the target, or promoting the production of the trispecific protein. In certain embodiments where it is desired to reduce effector function, the molecule is engineered to express a glycosylated form. In some embodiments, substitutions that result in elimination of one or more variable region framework glycosylation sites are included to eliminate glycosylation at the site. Conversely, in some cases, enhanced effector function or improved binding is conferred to the trispecific protein comprising Fc of the present disclosure by engineering at one or more additional glycosylation sites.

[0324] In some cases, the DLL3 targeting trispecific protein is differentially modified during or after production by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to an antibody molecule or other cellular ligand, etc. Any of a variety of chemical modifications are performed by techniques including, but not limited to, specific chemical cleavage by the following agents: cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

[0325] Various post-translational modifications also encompassed by the present disclosure include, for example, N-linked or O-linked carbohydrate chains, processing of the N-terminus or C-terminus, attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. In addition, in some cases, the DLL3 targeting trispecific binding protein is modified with a detectable label such as an enzyme, fluorescent, radioactive label, or affinity label to allow detection and isolation of the modulator.

[0326] Polynucleotide encoding DLL3 targeting trispecific protein

[0327] 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 in the form of DNA constructs. In other embodiments, the polynucleotide molecules are provided in the form of messenger RNA transcripts.

[0328] The polynucleotide molecules are constructed by known methods, for example by combining genes encoding three binding domains separated by a peptide linker, or in other embodiments directly linked by peptide bonds, into a single gene construct, which is operably linked to a suitable promoter and optionally a suitable transcription terminator, and expressed in bacteria or other appropriate expression systems, such as CHO cells. In embodiments where the DLL3 binding domain is a small molecule, the polynucleotide contains genes encoding a CD3 binding domain and a half-life extension domain. In embodiments where the half-life extension domain is a small molecule, the polynucleotide contains genes encoding domains that bind to CD3 and DLL3. Depending on the vector system and host used, any number of suitable transcription and translation elements may be used, including constitutive and inducible promoters. The promoter is selected so that it drives expression of the polynucleotide in the corresponding host cell.

[0329] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment. The recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, viruses (virii) (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc.) and cosmids.

[0330] A variety of expression vector / host systems can be used to contain and express the polynucleotide encoding the polypeptide of the trispecific antigen binding protein. Examples of expression vectors are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1): 111-27) for expression in E. coli, or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0331] Thus, in some embodiments, a DLL3 targeting trispecific protein as described herein is produced by introducing a vector encoding a protein as described above into a host cell and culturing the host cell under conditions allowing expression of the protein domain, which can be isolated and optionally further purified.

[0332] Pharmaceutical composition

[0333] In some embodiments, a pharmaceutical composition is also provided, comprising an anti-DLL3 trispecific binding protein as described herein, a vector comprising a polynucleotide encoding a polypeptide of the DLL3 targeting trispecific protein or a host cell transformed by the vector, and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the component and is non-toxic to the patient to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil-in-water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and can be administered to a subject at a suitable dose. Preferably, the composition is sterile. These compositions may also contain excipients such as preservatives, emulsifiers, and dispersants. Prevention of the action of microorganisms may be ensured by including various antibacterial and antifungal agents. Another embodiment provides one or more of the above-mentioned DLL3 targeting trispecific proteins, which are packaged in a lyophilized form or in an aqueous medium.

[0334] In some embodiments of the pharmaceutical composition, the DLL3 targeting trispecific protein described herein is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a fullerene, a liquid crystal, a liposome, a quantum dot, a superparamagnetic nanoparticle, a dendrimer, or a nanorod. In other embodiments of the pharmaceutical composition, the DLL3 targeting trispecific protein is attached to a liposome. In some cases, the DLL3 targeting trispecific protein is conjugated to the surface of the liposome. In some cases, the DLL3 targeting trispecific protein is encapsulated within the shell of the liposome. In some cases, the liposome is a cationic liposome.

[0335] The DLL3 targeting trispecific proteins described herein are contemplated for use as medicaments. Administration is achieved by different means, by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The dosing regimen will be determined by the attending physician based on other clinical factors. The dosage for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the specific compound to be administered, the time and route of administration, the type of therapy, general health, and other concurrently administered medications. An "effective dose" refers to the amount of active ingredient that is sufficient to affect the course and severity of the disease, thereby resulting in a reduction or alleviation of such pathology, and can be determined using known methods.

[0336] In some embodiments, the DLL3 targeting trispecific protein of the present disclosure is administered at a dose of up to 10 mg / kg at a frequency of once per week. In some cases, the dosage range is from about 1 ng / kg to about 10 mg / kg. In some embodiments, the dosage is from about 1 ng / kg to about 10 ng / kg, from about 5 ng / kg to about 15 ng / kg, from about 12 ng / kg to about 20 ng / kg, from about 18 ng / kg to about 30 ng / kg, from about 25 ng / kg to about 50 ng / kg, from about 35 ng / kg to about 60 ng / kg, from about 45 ng / kg to about 70 ng / kg, from about 65 ng / kg to about 85 ng / kg, from about 80 ng / kg to about 1 μg / kg, from about 0.5 μg / kg, from about 1 μg / kg to about 2 μg / kg, from about 2 μg / kg to about 3 μg / kg, from about 3 μg / kg to about 4 μg / kg, from about 5 μg / kg to about 6 μg / kg, from about 7 μg / kg to about 8 μg / kg, from about 9 μg / kg to about 10 μg / kg, from about 10 μg / kg to about 15 μg / kg, from about 12 ng / kg to about 20 ng / kg, from about 18 ng / kg to about 30 ng / kg, from about 25 ng / kg to about 50 ng / kg, from about 35 ng / kg to about 60 ng / kg, from about 45 ng / kg to about 70 ng / kg, from about 65 ng / kg to about 85 ng / kg, from about 80 ng / kg to about 1 μg / kg, from about 0.5 μg g / 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 dosage is about 0.1 mg / kg to about 0.2 mg / kg, about 0.25 mg / kg to about 0.5 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.75 mg / kg to about 3 mg / kg, about 2.5 mg / kg to about 4 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 6 mg / kg, about 5.5 mg / kg to about 7 mg / kg, about 6.5 mg / kg to about 8 mg / kg, about 7.5 mg / kg to about 9 mg / kg, or about 8.5 mg / kg to about 10 mg / kg. In some embodiments, the dosage is about less than once a day, once every other day, less than once a day, twice a week, once a week, once every 7 days, once every two weeks, once every two weeks, once every three weeks, once every four weeks, or once a month. In some cases, the dosage is once a week. In some cases, the dosage is once a week, and the dosage is up to 10 mg / kg. In some cases, administration duration is from about 1 day to about 4 weeks or longer.

[0337] Treatment

[0338] In some embodiments, the DLL3 binding protein or DLL3 targeting trispecific protein of the present disclosure is administered to treat a neoplastic condition. In some embodiments, the neoplastic condition is benign or malignant; solid tumor or other hematological tumor; and in some embodiments, selected from the group including, but not limited to, adrenal tumors, AIDS-related cancers, alveolar soft part sarcomas, astrocytic tumors, autonomic ganglion tumors, bladder cancer (squamous cell carcinoma and transitional cell carcinoma), blastocystic disorders, bone cancer (ameloid tumors, aneurysmal bone cysts, osteochondromas, osteosarcomas), brain and spinal cord cancer, metastatic brain tumors, breast cancer (including triple negative breast cancer), carotid body tumors, cervical cancer, chondrosarcomas, chordomas, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin, hyperplastic small round cell tumors, ependymomas, epithelial disorders, Ewing's tumors, extraosseous myxoid chondrosarcomas, incomplete fibroplasia of bone, fibrous fibrosis of bone dysplasia, gallbladder and bile duct cancer, gastric cancer, gastrointestinal cancer, gestational trophoblastic disease, germ cell tumors, glandular disorders, head and neck cancer, hypothalamus, intestinal cancer, islet cell tumors, Kaposi's sarcoma, kidney cancer (Wilms tumor, papillary renal cell carcinoma), leukemia, lipoma / benign lipoma, liposarcoma / malignant lipoma, liver cancer (hepatoblastoma, hepatocellular carcinoma), lymphoma, lung cancer (small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc.), macrophage disorders, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumors, pediatric cancer, peripheral nerve sheath tumors, pheochromocytoma, pituitary tumors, prostate cancer, posterious melanoma unveal melanoma), rare hematologic disorders, renal metastases, rhabdomyosarcoma, sarcomas, skin cancer, soft tissue sarcomas, squamous cell carcinoma, gastric cancer, stromal disorders, synovial sarcoma, testicular cancer, thymic cancer, thymoma, thyroid metastases, and uterine cancer (cervical, endometrial, and leiomyoma).

[0339] In certain embodiments, the DLL3 binding proteins or DLL3 targeting tri-specific proteins of the present disclosure are used as frontline therapy and are administered to subjects who have not previously received cancer treatment. In other embodiments, the DLL3 targeting tri-specific proteins of the present disclosure are used to treat subjects who have been previously treated (with the DLL3 targeting tri-specific proteins of the present disclosure or other anti-cancer agents) and have relapsed or are determined to be refractory to the previous treatment. In some embodiments, the DLL3 targeting tri-specific proteins of the present disclosure are used to treat subjects with recurrent tumors.

[0340] In some aspects, the DLL3 binding proteins or DLL3 targeting trispecific proteins of the disclosure are administered to treat proliferative disorders including solid tumors, including but not limited to carcinomas of the adrenal gland, liver, kidney, bladder, breast, stomach, ovary, cervix, uterus, esophagus, colorectal, prostate, pancreas, lung (small cell and non-small cell), thyroid, sarcomas, glioblastomas, and various head and neck tumors.

[0341] In some embodiments, a DLL3 binding protein or DLL3 targeting trispecific protein of the present disclosure is administered to a subject with melanoma. In some embodiments, a DLL3 targeting trispecific protein of the present disclosure is used to diagnose, monitor, treat or prevent melanoma. As used herein, the term "melanoma" includes all types of melanoma, including but not limited to primary melanoma, malignant melanoma, cutaneous melanoma, extracutaneous melanoma, superficial spreading melanoma, polyposis melanoma, melanoma, melanoepithelioma, melanosarcoma, melanoma in situ, nodular malignant melanoma, lentigo melanoma, lentigo maligna melanoma, mucosal lentigo melanoma, mucosal melanoma, acral melanoma, soft tissue melanoma, ocular melanoma, invasive melanoma, familial atypical mole and melanoma (FAM-M) syndrome, desmoplastic malignant melanoma or uveal melanoma.

[0342] DLL3 is an effective tumor marker that is expressed in a variety of different cancers and has been found to be associated with cancer stem cells. Therefore, in some embodiments where the DLL3 binding protein or DLL3 targeting trispecific protein of the present disclosure is incorporated into a chimeric antigen receptor expressed on a lymphocyte, the resulting "DLL3-sensitized lymphocyte" (e.g., a natural killer cell or a T cell that immunospecifically recognizes a DLL3 determinant) is able to effectively elicit an immune response against abnormal DLL3-positive cells (including cancer stem cells). This ability to effectively eliminate tumorigenic "seed" cells is often critical for reducing the likelihood of tumor recurrence or metastasis. In some embodiments, such DLL3-sensitized lymphocytes are used in combination with other therapeutic agents or are administered as part of a maintenance regimen following standard of care treatment.

[0343] More generally, a chimeric antigen receptor is an artificially constructed hybrid protein or polypeptide containing or comprising an antigen binding domain of an antibody connected to a signaling domain (e.g., a T cell signaling domain or a T cell activation 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) to DLL3-positive target cells in a non-MHC-restricted manner by utilizing the antigen binding properties of an antibody or its antigen-binding fragment. This non-MHC-restricted antigen recognition enables T cells expressing DLL3 CARs to recognize tumorigenic DLL3 independently of antigen processing, thereby bypassing the main mechanism of tumor escape. Moreover, when expressed in T cells, CAR does not favorably dimerize with endogenous T cell receptor (TCR) α and β chains.

[0344] In selected aspects, a DLL3 binding protein or DLL3 targeting trispecific protein of the present disclosure is incorporated into a chimeric antigen receptor (CAR), and the DLL3 CAR is administered in a CAR-based therapy that is effective in treating lung cancer, including the following subtypes: small cell lung cancer, non-small cell lung cancer (e.g., squamous cell non-small cell lung cancer or squamous cell small cell lung cancer), and large cell neuroendocrine carcinoma (LCNEC).

[0345] In some embodiments, DLL3 binding proteins or DLL3 sensitive lymphocytes are administered to patients exhibiting limited stage disease or extensive stage disease. In other embodiments, the disclosed DLL3 targeting trispecific antibodies are administered to refractory patients (i.e., patients whose disease relapses during or shortly after completing initial treatment); sensitive patients (i.e., patients whose relapse time is longer than 2-3 months after primary treatment); or patients who exhibit resistance to platinum drugs (e.g., carboplatin, cisplatin, oxaliplatin) and / or taxanes (e.g., docetaxel, paclitaxel, larotaxel, or cabazitaxel). In another embodiment, the disclosed DLL3 CAR therapy effectively treats ovarian cancer, including ovarian serous carcinoma and ovarian papillary serous carcinoma.

[0346] In some embodiments, the disclosed DLL3 binding proteins or DLL3 targeting trispecific binding proteins are used to prevent, treat, or diagnose tumors with neuroendocrine features or phenotypes, including neuroendocrine tumors. True or canonical neuroendocrine tumors (NETs) arising from the diffuse endocrine system are relatively rare, with an incidence of 2-5 cases per 100,000 people, but are highly aggressive. Neuroendocrine tumors occur in the kidneys, genitourinary tract (bladder, prostate, ovaries, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lungs (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 may be indicated by positive immunohistochemical markers such as neuron-specific enolase (NSE, also known as gamma enolase, gene symbol = ENO2), CD56 (or NCAM1), chromogranin A (CHGA), and synaptophysin (SYP), or genes known to be expressed at elevated levels such as ASCL1. Conventional chemotherapy is not particularly effective in treating neuroendocrine tumors, with liver metastases being a common outcome. In some embodiments, the disclosed DLL3-targeting trispecific antibodies are advantageously used to treat neuroendocrine tumors, and in some embodiments, they are used to treat, prevent, or diagnose pseudoneuroendocrine tumors (pNETs) that genotypically or phenotypically mimic, resemble, or exhibit common features with standard neuroendocrine tumors. Pseudoneuroendocrine tumors, or tumors with neuroendocrine features, arise from cells of the diffuse neuroendocrine system or from cells in which the neuroendocrine differentiation cascade has been aberrantly reactivated during carcinogenesis. Such pNETs share certain phenotypic or biochemical characteristics with traditionally defined neuroendocrine tumors, including the ability to produce a subset of bioactive amines, neurotransmitters, and peptide hormones. Histologically, such tumors (NET and pNET) have a common appearance, typically showing small cells with tight junctions, cytoplasm with minimal mild cytopathology, and round to oval punctate nuclei. In some embodiments of the present disclosure, the commonly expressed histological markers 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). Therefore, in some embodiments, the DLL3-targeted trispecific protein, DLL3CAR or DLL3-sensitized lymphocytes of the present disclosure, or any combination thereof, are beneficially used to treat pseudo-neuroendocrine tumors and canonical neuroendocrine tumors, for example, for the treatment of neuroendocrine tumors (NET and pNET) occurring in the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lung (small cell lung cancer and large cell neuroendocrine carcinoma).In addition, in some embodiments, the DLL3 targeting tri-specific protein, DLL3 CAR or DLL3 sensitized lymphocyte of the present disclosure, or any combination thereof, is used to treat tumors expressing one or more markers, such as NSE, CD56, synaptophysin, chromogranin A, ASCL1 or PGP9.5 (ETCHL1). In some embodiments, the DLL3 targeting tri-specific protein, DLL3 CAR or DLL3 sensitized lymphocyte of the present disclosure, or any combination thereof, is used to treat subjects with tumors that are NSE+ or CD56+ or PGP9.5+ or ASCL1+ or SYP+ or CHGA+ or any combination thereof.

[0347] In another embodiment, the DLL3 targeting tri-specific protein, DLL3 CAR or DLL3-sensitized lymphocytes of the present disclosure, or any combination thereof, are used in maintenance therapy to reduce or eliminate the chance of tumor recurrence after the initial appearance of the disease. In some cases, the disease has been treated and the initial tumor mass has been eliminated, reduced or otherwise improved, so that the patient is asymptomatic or in a state of remission. At this time, a pharmaceutically effective amount of the disclosed DLL3 binding protein, the DLL3 targeting tri-specific protein of the present disclosure, DLL3 CAR or DLL3-sensitized lymphocytes, or any combination thereof, is administered to the subject once or more, regardless of whether there are few or no disease indications using standard diagnostic procedures. In some embodiments, the DLL3 targeting tri-specific protein, DLL3 CAR or DLL3-sensitized lymphocytes of the present disclosure, or any combination thereof, is administered on a regular schedule, such as weekly, biweekly, monthly, every six weeks, every two months, every three months, every six months or once a year over a period of time to reduce the possibility of disease recurrence. In addition, in some embodiments, depending on the patient's response and clinical and diagnostic parameters, this treatment continues for weeks, months, years, or even indefinitely.

[0348] In yet another embodiment, the DLL3 binding protein, the DLL3 targeting tri-specific protein of the present disclosure, the DLL3 CAR or the DLL3 sensitized lymphocytes or any combination thereof is used for preventive or adjuvant therapy following a reduction program to prevent or reduce the likelihood of tumor metastasis. As used in the present disclosure, a "reduction program" is broadly defined and means any procedure, technique or method to eliminate, reduce, treat or improve tumors or tumor proliferation. Exemplary reduction programs include, but are not limited to, surgery, radiotherapy (i.e., beam radiation), chemotherapy, immunotherapy or ablation. In some embodiments, at an appropriate time, as recommended by a clinical, diagnostic or therapeutic method, a DLL3 binding protein, a DLL3 targeting tri-specific protein of the present disclosure, a DLL3 CAR or a DLL3 sensitized lymphocyte or any combination thereof is administered to reduce tumor metastasis. In some embodiments, the dosing regimen is accompanied by appropriate diagnostic or monitoring techniques that allow it to be modified.

[0349] Other embodiments of the present disclosure include administering a DLL3 binding protein, a DLL3 targeting tri-specific protein of the present disclosure, a DLL3 CAR, or a DLL3-sensitized lymphocyte, or any combination thereof, to a subject who is asymptomatic but at risk of developing a proliferative disease. That is, in some embodiments, a DLL3 binding protein, a DLL3 targeting tri-specific protein of the present disclosure, a DLL3 CAR, or a DLL3-sensitized lymphocyte, or any combination thereof, is used in a preventive sense and provided to a patient who has been examined or tested and has one or more noteworthy risk factors (e.g., genomic indications, family history, in vivo or in vitro test results, etc.), but has not yet developed a tumor. In such a case, a person skilled in the art will be able to determine an effective dosing regimen by empirical observation or by recognized clinical practice.

[0350] In some embodiments, as used herein, "treatment" or "treatment" refers to therapeutic treatment, wherein the purpose is to slow down (mitigate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reduction in the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the condition, disorder or disease state; delayed onset or slowed progression of the condition, disorder or disease; improvement of the condition, disorder or disease state; and detectable or undetectable relief (partial or complete) or enhancement or improvement of the condition, disorder or disease. Treatment includes causing a clinically significant response without excessive levels of side effects. Treatment also includes extending survival compared to the expected survival without treatment. In other embodiments, "treatment" or "treatment" refers to preventive measures, wherein the purpose is, for example, in a person susceptible to the disease (e.g., an individual carrying genetic markers for a disease such as breast cancer), delaying the onset of an undesirable physiological condition, disorder or disease or reducing its severity.

[0351] In some embodiments of the methods described herein, a DLL3 binding protein, DLL3 targeting trispecific protein, or composition as described herein is administered in combination with an agent used to treat a particular disease, disorder, or condition. Agents include, but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutic drugs), hormones (steroids, peptides, etc.), radiation therapy (gamma rays, X-rays, and / or targeted delivery of radioisotopes, microwaves, UV radiation, etc.), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapies. In some embodiments, an anti-DLL3 binding protein or anti-DLL3 targeting trispecific protein described herein is administered in combination with an antidiarrheal, antiemetic, analgesic, opioid, and / or nonsteroidal anti-inflammatory agent. In some embodiments, an anti-DLL3 binding protein or anti-DLL3 targeting trispecific protein described herein is administered in combination with an anticancer agent. Non-limiting examples of anticancer agents that can be used in various embodiments of the present disclosure, including pharmaceutical compositions and dosage forms and kits of the present disclosure, include: acivicin; aclarubicin; acodazole hydrochloride; aclonine; adolesin; aldesleukin; hexamethylmelamine; ambomycin; ametrine acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; tricline; azacitidine; azatepa; azomycin; batimastat; Benzotepa; Bicalutamide; Bisantrene hydrochloride; Bisantrene dimesylate; Bizeresin; Bleomycin sulfate; Buquina sodium; Bropiridamine; Busulfan; Actinomycin C; Carotene; Caracetamide; Carbetim; Carboplatin; Carmustine; Carrubicin hydrochloride; Carzeresin; Cedifenol; Chlorambucil; Ciromycin; Cisplatin; Cladribine; Crestor mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Actinomycin D; Daunorubicin hydrochloride decitabine; dextromaplatin; dezaguanine; dezaguanine mesylate; diazurone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; drolostanone propionate; dazomicin; edatrexamate; eflornithine hydrochloride; elsamitrucin; enloplatin; enprobamate; epipipridine; epirubicin hydrochloride; erbulazole; esorubicin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate cefoside; etobanim; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flucitabine; fosquatone; fostramexine sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; imofosine; interleukin II (including recombinant interleukin II or rIL2); interferon α-2a; interferon α-2b; interferon α-n1; interferon α-n3; interferon β-I a; interferon γ-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; lirazole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprofen; maytansine; nitrogen mustard hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menorel; mercaptopurine; methotrexate;Methotrexate sodium; chlorpheniramine; metodepa; mibutidamide; mitocarcin; mitoclomin; mitocrin; mitomycin; mitospe; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogamycin; ormaplatin; oxysulen; paclitaxel; pegaspargase; pelimycin; pentamidine; pelimycin sulfate; perfosfamide; piperobroman; piperosulfan; pyroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazolidinone; riboadenylate; roglulimide; safengo; safengo hydrochloride; semustine; sphenobarbital; spardronate sodium; sparmycin; salt Spirogermanium acid; spiromustine; spiroplatin; streptozocin; streptozocin; sulfochlorophenylurea; talithromycin; tecogalan sodium; tegafur; tiloxantrone hydrochloride; temoporfin; teniposide; tiroxilon; testolactone; thiamidine; thioguanine; thiotepa; thiazolamide nucleoside; tirapazamine; toremifene citrate; triptolon acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronide; triptorelin; tobradazole hydrochloride; uramustine; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinpicrine sulfate; vinpoxetine sulfate; vinorelbine sulfate; vinorelbine tartrate; vinprodine sulfate; vinblastine sulfate; vorozole; zeniplatin; nestatin; zorubicin hydrochloride. Other examples of anticancer drugs include, but are not limited to, 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adenosine; adolesin; aldesleukin; ALL-TK antagonists; hexamethylmelamine; aminomustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; anrelix; anti-dorsalizing morphogenetic protein-1 (anti-dorsalizing morphogenetic protein-1); protein-1); antiandrogen; prostate cancer; antiestrogens; antineoplastons; antisense oligonucleotides; glycine aphidicolin; apoptosis gene regulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; amustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; diazotyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; β-lactam derivatives; β-alethine; betaclamycin B; betulinic acid; bFGF inhibitors;bicalutamide; bisantrene; diaziridinylspermine; binefad; bistratene A; bisezolesine; breflate; bropiridamine; budotetan; buthionine sulfate imide; calcipotriol; calciphosphotin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamide triazole; CaRest M3; CARN 700; cartilage-derived inhibitor; carzolesine; casein kinase inhibitor (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomiphene analogs; clotrimazole; collismycin A; collismycin B; Conagenin A4; Conagenin analog; conagenin; crambescidin 816; Crestor; Candida albicans 8; Candida albicans A derivative; curacin A; cyclopentanthrone; cycloplatam; sepimycin; cytarabine octadecyl phosphate; cytolytic factor; estradiol phosphate (cytostatin); dacilizumab; decitabine; dehydrocannabinol B; deslorelin; dexamethasone; right ifosfamide; dexrazoxane; right verapamil; diazocone; candida albicans B; didox; diethylnorspermine; dihydro-5-azacytidine; 9-dihydrotaxol; dioxamycin cin); diphenylspiromustine; docetaxel; docosinol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edilfosine; edrecolomab; eflornithine; elemene; ethiprofen; epirubicin; aprelet; estramustine analogs; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim ; finasteride; flavopiridol; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; fofolmec; formestane; fostramoxine; fostramustine; gadoliniumtexaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; egg-tonic white; hexamethylenebisacetamide; hyperforin; ibandronic acid; idarubicin; idoxifene; idarucizumab; imofosine; ilomastat; imidazoacridone; imiquimod; immunostimulatory peptides; insulin-like growth factor-I receptor inhibitors; interferon agonists; interferons; interleukins; iodobenzylguanidine; iododexorubicin; 4-adenosine; iropra; irsogladine; isobongazole; isohomohalicongol B; itasetron; jasplakinolide;Kahalalide F; spirochetin triacetate-N; lanreotide; ranamycin; levograstim; lentinan sulfate; rebostatin; letrozole; leukemia inhibitory factor; interferon alpha; leuprolide + estrogen + progesterone; leuprolide; levamisole; lirazole; linear polyamine analogs; lipophilic disaccharide peptides; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; earthworm phospholipids; lometrexol; lonidamine; losoxantrone; HMG-CoA reductase inhibitors (such as but not limited to lovastatin, pravastatin, fluvastatin, statins, simvastatin and atorvastatin); loxoribine; lutetium texaphyrin; lysofylline; lytic peptide; maytansine; manostatin A; marimastat; masoprofen; mammoplastin; matrix lytic factor inhibitor; matrix metalloproteinase inhibitor; menolipin; merbarone; avorelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; miriplastine; mismatched double-stranded RNA; mitoxantrone; dibromodulanol; mitomycin analog; mitonadamide; metotoxin fibroblast growth factor-saporin; mitoxantrone; mofaroten; morasplastine; human chorionic gonadotropin monoclonal antibody; monophosphoryl lipid A+ mycobacterial cell wall sk; mopidarol; multidrug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; Indian Ocean sponge B (mycaperoxide B); mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; narostem; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidants; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducers; ormaplatin; osetaxelone; oxadiazine oxaunomycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronate; panaxatriol; panomifen; parabactin; pozeptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentozole; perflurane; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; pyrexine; placetin A; placetin B;plasminogen activator inhibitors; platinum complexes; platinum compounds; platinum-triamine complexes; porfimer sodium; porfiromycin; prednisone; propyl diacridone; prostaglandin J2; proteasome inhibitors; protein A-based immunomodulators; protein kinase C inhibitors; protein kinase C inhibitors; microalgae; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurin; methoxypyrazoline acridinium; pyridoxal hemoglobin polyoxyethylene conjugates; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; demethylated reteptine; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII isotretinoin; roglulimide; roxitomine; romotide; roquineme; rubiginone B1; ruboxyl; safengo; saintopin; SarCNU; phytol A; sargramostim; Sdi 1 mimetic; semustine; senescence-derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction regulator; single-chain antigen binding protein; sizoran; sobuzoxane; borcarbazine sodium; sodium phenylacetate; solverol; somatomedin binding protein; sonamyl; fospartate; spicamycin D; spiromustine; spongistatin 1 1); squalamine; stem cell inhibitor; stem cell division inhibitor; stipiamide; stromelysin inhibitor; sulfinosine; potent vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycan; tamustine; tamoxifen methylthiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitor; temoporfin; temozolomide; teniposide; tetrachlorodecaoxid; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymofasin; thymopoietin receptor agonist; thymosin; thyroid stimulating hormone; tin ethyletiopurpurin; tirapazamine; titanocene dichloride bichloride); topsentin; toremifene; totipotent stem cell factor; translation inhibitor; retinoic acid; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turotelene; tyrosine kinase inhibitor; tyrosine phosphorylation inhibitor (tyrphostin); UBC inhibitor; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonist; vapreotide; variolin B; vector system; erythrocyte gene therapy; veraresol; veratramine; verdins;Verteporfin; Vinorelbine; Vinxaltine; Vorozole; zanoteron; zeniplatin; zilascorb; and nestatins. Other anticancer drugs are 5-fluorouracil and folinic acid. These two agents are particularly useful when used in a method using thalidomide and a topoisomerase inhibitor. In some embodiments, the DLL3-targeting trispecific proteins of the present disclosure are used in combination with gemcitabine. In some embodiments, the DLL3-targeting trispecific proteins described herein are administered before, during, or after surgery.

[0352] Detection methods of DLL3 expression and diagnosis of DLL3-related cancers

[0353] 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 containing a labeled anti-DLL3 single domain antibody or antigen-binding fragment thereof), and one or more compounds for detecting a marker. In some embodiments, the marker is selected from a fluorescent marker, an enzyme marker, a radioactive marker, a nuclear magnetic resonance active marker, a luminescent marker, and a chromophore marker.

[0354] In some cases, DLL3 expression is detected in a biological sample. The sample can be any sample, including but not limited to tissues from biopsies, autopsies, and pathology specimens. Biological samples also include tissue sections, e.g., frozen sections for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates.

[0355] In one embodiment, a method of determining whether a subject has cancer is provided by contacting a sample from the subject with an anti-DLL3 single domain antibody or an anti-DLL3 tri-specific protein disclosed herein and detecting binding of the single domain antibody to the sample. An increase in binding of the antibody to the sample compared to binding of the antibody to a control sample determines that the subject has cancer.

[0356] In another embodiment, a method of confirming the diagnosis of cancer in a subject is provided by contacting a sample from a subject diagnosed with cancer with an anti-DLL3 single domain antibody or an anti-DLL3 tri-specific protein disclosed herein and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample confirms the diagnosis of cancer in the subject.

[0357] In some examples of the disclosed methods, 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 binding of the second antibody. Increased binding of the second antibody to the sample as compared to binding of the second antibody to a control sample detects cancer in the subject or confirms a diagnosis of cancer in the subject. In some cases, the cancer is a neuroendocrine cancer, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, triple negative breast cancer or ovarian cancer, or any other type of cancer that expresses DLL3. In some examples, the control sample is a sample from a subject without cancer. In specific examples, the sample is a blood or tissue sample.

[0358] In some cases, the antibody that binds (e.g., specifically binds) DLL3 is directly labeled with a detectable marker. In another embodiment, the antibody that binds (e.g., specifically binds) DLL3 (the first antibody) is not labeled, and the second antibody or other molecule that can bind to the antibody that specifically binds DLL3 is labeled. The second antibody is selected so that it can specifically bind to a particular class and category of the first antibody. For example, if the first antibody is llama IgG, the second antibody can be anti-llama IgG. Other molecules that can bind to the antibody include, but are not limited to, protein A and protein G, both of which are commercially available. Suitable labels for the antibody or the second antibody are described above and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group 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.

[0359] In an alternative embodiment, DLL3 can be measured in a biological sample by a competitive immunoassay that employs a DLL3 standard labeled with a detectable substance and an unlabeled antibody that specifically binds DLL3. In this assay, the biological sample, the labeled DLL3 standard, and the antibody that specifically binds DLL3 are mixed together, and the amount of the labeled DLL3 standard bound to the unlabeled antibody is measured. The amount of DLL3 in the biological sample is inversely proportional to the amount of the labeled DLL3 standard bound to the antibody that specifically binds DLL3.

[0360] The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, an antibody that specifically binds DLL3 can be used to detect the production of DLL3 in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of DLL3 in a biological sample, such as a tissue sample or a blood or serum sample. In some examples, the DLL3 is cell surface DLL3. In other examples, the DLL3 is soluble DLL3 (e.g., DLL3 in a cell culture supernatant or soluble DLL3 in a body fluid sample, such as a blood or serum sample.

[0361] In one embodiment, a kit for detecting DLL3 in a biological sample, such as a blood sample or a tissue sample, is provided. For example, to confirm a cancer diagnosis in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample can be obtained to detect the presence of a soluble DLL3 protein or fragment. According to the present disclosure, a kit for detecting a polypeptide will generally contain a single domain antibody that specifically binds DLL3. In some embodiments, an antibody fragment, such as a scFv fragment, a VH domain, or a Fab, is included in the kit. In further embodiments, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzymatic marker).

[0362] In one embodiment, the kit includes instructional material that discloses means for using an antibody that binds DLL3. The instructional material may be written, electronic (e.g., on a computer diskette or compact disk), visual (e.g., a video file), or provided via an electronic network, such as the Internet, the World Wide Web, an intranet, or other network. The kit may also include other components to facilitate the specific application for which the kit is designed. Thus, for example, the kit may additionally include means for detecting the marker (e.g., an enzyme substrate for an enzyme marker, a filter set for detecting a fluorescent marker, a suitable secondary marker, such as a secondary antibody, etc.). The kit may additionally include buffers and other reagents conventionally used to practice a particular method. Such kits and appropriate contents are well known to those skilled in the art.

[0363] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassay may vary depending on the particular format employed, methods for detecting DLL3 in a biological sample generally include the steps of contacting the biological sample with an antibody that specifically reacts with a DLL3 polypeptide under immunoreactive conditions. The antibody is allowed to specifically bind under immunoreactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0364] Methods for determining the presence or absence of cell surface markers are well known in the art. For example, antibodies can be conjugated to other compounds including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorescent dyes, metal compounds, radioactive compounds, or drugs. Antibodies can also be used in immunoassays, such as, but not limited to, radioimmunoassays (RIA), ELISA, or immunohistochemistry assays. Antibodies can also be used in fluorescence activated cell sorting (FACS). FACS uses multiple color channels, low-angle and obtuse light scatter detection channels, and impedance channels, as well as other more complex detection levels, to separate or classify cells (see U.S. Pat. No. 5,061,620). As disclosed herein, any single domain antibody that binds to DLL3 can be used in these assays. Therefore, these antibodies can be used in conventional immunoassays, including, but not limited to, ELISA, RIA, FACS, tissue immunohistochemistry, Western blotting, or immunoprecipitation.

[0365] Example

[0366] Example 1: Screening of phage display libraries for identification of DLL3 binding domains

[0367] Used in EXPI293 TM Llamas were immunized with purified DLL3 protein expressed in cells. Phage display libraries for expressing heavy chain variable antibody domains were constructed from circulating B cells (see van der Linden, de Geus, Stok, Bos, van Wassenaar, Verrips, and Frenken. 2000. J Immunol Methods 240: 185–195). Phage clones were screened for binding to DLL3 by expressing the clones in E. coli, preparing periplasmic extracts, and screening the clones for DLL3 binding activity by ELISA. Fifty-two unique heavy chain-only single domain antibodies were identified that produced a signal in the ELISA screen (SEQ ID Nos. 1 to 52). The CDR1, CDR2, and CDR3 sequences of these heavy variable domains are SEQ ID Nos. 443 to 494, SEQ ID Nos. 885 to 936, and SEQ ID Nos. 1327 to 1378, respectively.

[0368] Example 2: Humanization of DLL3-binding single domain antibodies and T cell-dependent cytotoxicity assays

[0369] Thirty-four (SEQ ID Nos. 53 to 86) exemplary llama anti-DLL3 heavy chain only single domain antibodies from Example 1 were humanized. The CDR1, CDR2 and CDR3 sequences of these 34 heavy chain only single domain antibodies are SEQ ID Nos. 495 to 528, SEQ ID Nos. 937 to 970 and SEQ ID Nos. 1379 to 1412, respectively.

[0370] The humanized anti-DLL3 sequence was cloned into an expression vector comprising the signal domain followed by the anti-DLL3 heavy chain only variable domain followed by a GGGGSGGGS linker (SEQ ID No. 1808), followed by the anti-human albumin domain antibody 10G (SEQ ID No. 1774), followed by a GGGGSGGGS linker (SEQ ID No. 1808), followed by the anti-human CD3 antibody 2B2 (SEQ ID No. 1793), followed by a HHHHHH tag (SEQ ID No. 1819), thereby generating an anti-DLL3 trispecific construct.

[0371] The anti-DLL3 trispecific construct containing the humanized anti-DLL3 binding sequence was then transfected into EXPI293 TM These anti-DLL3 trispecific constructs were engineered with protein A binding sites and transfected EXPI293 cells were quantified using an Octet instrument with a protein A tip. TM The amount of anti-DLL3 trispecific construct in the conditioned medium of the cells was quantified. A trispecific protein of similar molecular weight to the anti-DLL3 trispecific protein was used as a standard.

[0372] Using conditioned medium containing known concentrations of anti-DLL3 trispecific proteins, the binding affinity of anti-DLL3 trispecific proteins to human and cynomolgus monkey DLL3 proteins was measured using the following method in which the DLL3 proteins were expressed as human IgG1-Fc fusions and measured using an Octet instrument with an anti-human Fc tip. K was measured using a single 50 nM concentration of anti-DLL3 trispecific proteins. D The relative affinities measured as described above are listed in Table 1. All sequences were found to bind with relative affinities (K) ranging from 0.5 to 42 nM. D ) binds to human DLL3. Some sequences were found to bind to cynomolgus monkey DLL3 with similar affinity to human DLL3, and the relative affinity of these sequences for binding to cynomolgus monkey DLL3 is also shown in Table 1.

[0373] Conditioned medium was also tested by 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. CRL-2064 TM ) were combined with purified human T cells from donors and titrated with the anti-DLL3 trispecific proteins tested.

[0374] Hypothetically, if the anti-DLL3 trispecific protein directs T cells to kill DMS-153 cells expressing DLL3, the viability of DMS-153 cells as determined by luciferase assay 48 hours after the start of the experiment should be reduced.

[0375] Figure 2-6 Graphs showing representative TDCC data showing that several exemplary anti-DLL3 trispecific proteins were able to reduce the viability of DMS-153 cells. Figure 2 Shown are the results of a TDCC assay against an anti-DLL3 trispecific protein comprising the DLL3 binding domains DH18 (SEQ ID No. 59), DH11 (SEQ ID No. 55), DH67 (SEQ ID No. 42) and DH56 (SEQ ID No. 73). Figure 3 Shown are the results of a TDCC assay against an anti-DLL3 trispecific protein comprising the DLL3 binding domains DH2 (SEQ ID No. 60), DH43 (SEQ ID No. 68), DH10 (SEQ ID No. 54) and DH6 (SEQ ID No. 75). Figure 4 Shown are the results of a TDCC assay for a DLL3 trispecific protein comprising the DLL3 binding domains DH82 (SEQ ID No. 81), DH23 (SEQ ID No. 62), DH89 (SEQ ID No. 84) and DH17 (SEQ ID No. 58). Figure 5 Shown are the results of a TDCC assay for a DLL3 trispecific protein comprising the DLL3 binding domains DH83 (SEQ ID No. 82), DH12 (SEQ ID No. 56), DH61 (SEQ ID No. 76) and DH29 (SEQ ID No. 64). Figure 6The results of the TDCC assay of a DLL3 trispecific protein comprising the DLL3 binding domains DH58 (SEQ ID No. 74) and DH70 (SEQ ID No. 79) are shown. The negative control for the TDCC assay was a trispecific protein targeting GFP instead of DLL3 (e.g. Figure 6 Table 1 also lists the EC values ​​obtained by TDCC assay. 50 These values ​​ranged from 69 pM to 11 nM.

[0376] Table 1: Activity of humanized anti-DLL3 trispecific proteins in the DMS-153TDCC assay and their affinity for human and cynomolgus monkey DLL3 proteins. K values ​​were determined using a single concentration of anti-DLL3 trispecific proteins. D Measurements. TDCC assay using human T cells. n / d indicates no binding detected.

[0377]

[0378] Example 3: Phage display of the vector using two humanized DLL3 single domain antibodies from previous examples Screening of libraries to identify DLL3 binding domains with higher binding affinity

[0379] Two humanized antibody sequences, DH43 (SEQ ID No. 68) and DH6 (SEQ ID No. 75), were used as starting points for the preparation of phage display libraries (according to WO2016187101A2). The anti-DLL3 sequences from this panning were then cloned into an expression vector, with the expression construct comprising a signal domain, followed by the heavy chain-only anti-DLL3 variable domain, followed by a GGGGSGGGS linker (SEQ ID No. 1808), followed by an anti-human albumin single domain antibody domain, followed by a GGGGSGGGS linker (SEQ ID No. 1808), followed by an anti-human CD3 antibody fragment, and then a HHHHHH tag (SEQ ID No. 1819) to produce an anti-DLL3 trispecific protein. These constructs were transfected into EXPI293 TMcells, 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 to 367. Table 2 provides the CDR changes obtained in the DH43 DLL3 binder sequences after phage display selection. Three clones identified from the panning, SEQ ID Nos. 199 (2E05), 330 (4D09), and 365 (4H011), were engineered to generate variants, wherein each variant had a single amino acid change compared to the parent sequence, for example, to remove potential metabolic liabilities of the parent sequence. In particular, the DLL3 binding domains comprising SEQ ID No. 227 (2E05-M106Y), 228 (2E05-M106Q) are engineered variants of SEQ ID No. 199 (2E05); SEQ ID No. 366 (4D09-M34L) is an engineered variant of SEQ ID No. 330 (4D09); SEQ ID No. 367 (4H11-M34L) is an engineered variant of SEQ ID No. 365 (4H011). The CDR1 sequences of these DLL3 binding clones identified by panning are SEQ ID No. 529 to 809, the CDR2 sequences of clones identified by panning are SEQ ID No. 971 to 1251, and the CDR3 sequences of clones identified by panning are SEQ ID No. 1413 to 1691.

[0380] Table 2: Variations in CDR sequences of DH43 and its derivatives by amino acid position

[0381]

[0382]

[0383] The binding affinity of the anti-DLL3 trispecific protein to human DLL3 protein was measured using conditioned medium with known concentrations of the anti-DLL3 trispecific protein, using a method in which the biotinylated form of the human DLL3 protein was expressed as a human IgG1 fusion protein, and the binding affinity was determined in an Octet instrument with a streptavidin tip. K was measured using a single 50 nM concentration of the anti-DLL3 trispecific protein. D measurement, which allows ranking of potency. In this experiment, the relative K D The values ​​ranged from 2.3 nM to 64 nM, as shown in Table 3. Based on four samples of conditioned media from four transfections, the parent binders DH43 and DH6 had K values ​​of 7.7 ± 0.6 nM and 9.9 ± 0.3 nM, respectively. D value.

[0384] For the specific DLL3 binder molecules identified in this round of panning, as well as the parental DLL3 binders DH43 and DH6, more precise affinity measurements against 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 proteins. The binding affinities determined by more precise measurements of certain 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 study, the K of the parent binder DH43 was D The K value of the parent binder DH6 was 8.9 nM, while the affinity of the sub-molecule with the highest affinity, 1H012 (SEQ ID No. 162), was 2.9 nM. In addition, 1H012 (SEQ ID No. 162) also retained the ability to bind to cynomolgus monkey DLL3. D The value is 9.0 nM, while the affinity of the sub-molecule with the highest affinity, 4H011 (SEQ ID No. 365), is 3.9 nM. In addition, 4H011 (SEQ ID No. 365) also retains the ability to bind to cynomolgus monkey DLL3.

[0385] Using the same protocol as described in Example 2, 22 DLL3 binder molecules identified in this round of panning were selected for testing in the TDCC assay using DMS-153 cells. Exemplary TDCC data are provided in Figure 7-11 The EC 50 Summary of values. In this experiment, the EC values ​​of the parental DLL3 molecules DH43 and DH6 were 50 The most effective daughter molecule of DH43 is 1H012 (SEQ ID No. 162), with an EC 50 The value was 28 nM, showing a 7-fold increase in TDCC potency compared to the parental DLL3 binder DH43. The most potent daughter molecule of DH6 was 4H011 (SEQ ID No. 365), with an EC 50The value was 36 nM, thus an 8-fold increase in TDCC potency compared to the parental DLL3 binder molecule. A control trispecific protein targeting GFP used as a control had no activity in this assay ( Fig.11 shown).

[0386] Table 3: Relative affinities of anti-DLL3 trispecific proteins

[0387]

[0388]

[0389] Table 4: Binding constants for human DLL3 determined using three different concentrations of anti-DLL3 trispecific proteins and for cynomolgus monkey DLL3 determined using a single concentration of anti-DLL3 trispecific proteins

[0390]

[0391] Table 5: DMS-153 TDCC values ​​for affinity matured anti-DLL3 trispecific proteins in conditioned media tested in triplicate using human T cells

[0392]

[0393]

[0394] Example 4: Cloning of selected DLL3 binding molecules from Example 3 into mammals

[0395] The anti-DLL3 trispecific protein and the parental DLL3 binding molecule described in Example 3 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 binding molecules are: 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). After expression of the anti-DLL3 trispecific protein in CHO cells, it was purified from the conditioned medium of the stable clone pool using protein A and ion exchange chromatography. The purified protein was tested in the TDCC assay using the same method as described in Example 2. The EC values ​​from the TDCC assay of this example are listed in Table 6 50 values, and the data is graphically presented in Figure 12-15 The EC of the most effective molecule 2E05-M106Q (SEQ ID No. 228) is given in 50 The value was 41 nM, which is 6.6 times that of its parent molecule DH43. The most effective molecule derived from DH6 is 4D09-M34L (SEQ ID No. 366), with an EC 50 The value was 54 nM, which was 4.4 times more potent than the parent molecule DH6.

[0396] Table 6: TDCC activity of CHO expressed and purified affinity matured anti-DLL3 trispecific proteins

[0397]

[0398]

[0399] Example 5: Affinity maturation to obtain anti-DLL3 binders with improved affinity

[0400] In order to obtain more potent anti-DLL3 binders, a second round of affinity maturation was performed. A phage display library was created based on the DH6 (SEQ ID No. 75) and DH58 (SEQ ID No. 74) parental sequences. Sequences of binders from this round of affinity maturation are provided by SEQ ID Nos. 368 to 442. The CDR1 sequences of DLL3 binders identified in this round of affinity maturation are SEQ ID Nos. 810 to 884, the CDR2 sequences of DLL3 binders identified in this round of affinity maturation are SEQ ID Nos. 1252-1326, and the CDR3 sequences of DLL3 binders identified in this round of affinity maturation are SEQ ID Nos. 1692 to 1768. Table 7 provides the CDR changes obtained in the DH6 DLL3 binding sequences after phage display selection.

[0401] The affinity matured anti-DLL3 sequence identified as described above was cloned into an expression vector comprising a signal domain followed by the anti-DLL3 sequence followed by a GGGGSGGGS linker (SEQ ID No. 1808), followed by the anti-human albumin single domain antibody 10G (SEQ ID No. 1774), followed by a GGGGSGGGS linker (SEQ ID No. 1808), followed by the anti-human CD3 antibody 2B2 (SEQ ID No. 1793), followed by a HHHHHH tag (SEQ ID No. 1819), to generate an anti-DLL3 trispecific construct.

[0402] The anti-DLL3 trispecific construct containing the affinity-matured anti-DLL3 binding sequence was then transfected into EXPI293 TM These anti-DLL3 trispecific constructs were subsequently engineered with protein A binding sites and transfected EXPI293 cells were cloned using an Octet instrument with a protein A tip. TM The amount of anti-DLL3 trispecific construct in the conditioned medium of the cells was quantified. A control trispecific protein with a molecular weight similar to the anti-DLL3 trispecific protein was used as a standard.

[0403] The relative binding affinity of the anti-DLL3 trispecific protein to human DLL3 protein was measured using conditioned medium with known concentrations of the anti-DLL3 trispecific protein, using a method in which a biotinylated form of the human DLL3 protein was expressed as a human IgG1 fusion protein, and binding affinity measurements were performed in an Octet instrument with a streptavidin tip. K was measured using a single 50 nM concentration of the anti-DLL3 trispecific protein. D Affinities were measured, which allowed for ranking of potency. The measured affinities are listed in Table 8. All sequences tested were found to bind human DLL3, KD The values ​​ranged from 0.3 nM to 34 nM.

[0404] Conditioned media were also tested by a T cell-dependent cytotoxicity assay (see Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27). In this assay, luciferase-labeled DMS-153 cells were combined with purified human T cells and titrated anti-DLL3 trispecific proteins. It was hypothesized that if the anti-DLL3 trispecific protein directed T cells to kill DMS-153 cells expressing DLL3, the viability of DMS-153 cells determined by the luciferase assay 48 hours after the start of the experiment should be reduced. Fig.16 A graph showing 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), and for purified DH43 protein (SEQ ID No. 68) and purified DH6 protein (SEQ ID No. 75). Table 9 lists the EC values ​​obtained by TDCC assay. 50 The values ​​ranged from 4.2 pM to 1.5 nM. The negative control used for the TDCC assay was a trispecific protein targeting GFP (e.g. Fig.16 ), which does not direct T cells to kill DMS-153 cells.

[0405] Table 7: Variations in CDR sequences of DH6 and its derivatives by amino acid position

[0406]

[0407] Table 8: Binding constants for human DLL3 determined using a single concentration of anti-DLL3 trispecific proteins

[0408]

[0409]

[0410]

[0411]

[0412] Table 9: DMS-153 TDCC values ​​for affinity matured anti-DLL3 trispecific proteins in conditioned media tested in triplicate using human T cells

[0413]

[0414]

[0415]

[0416] Example 6: Affinity maturation to obtain anti-DLL3 binders with improved affinity

[0417] Certain anti-DLL3 trispecific proteins containing the DLL-3 binding sequences with the strongest TDCC activity in the assay described in Example 5 and anti-DLL3 trispecific proteins containing the parental DLL3 binder DH6 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 binding sequences are: DH6 (SEQ ID No. 75); 51A2 (SEQ ID No. 408); 51A5 (SEQ ID No. 411); 51F3 (SEQ ID No. 423); 51G2 (SEQ ID No. 425); 51G10 (SEQ ID No. 427); 51H5 (SEQ ID No. 429); 51X5 (SEQ ID No. 1886); 52B1 (SEQ ID No. 430); 52C4 (SEQ ID No. 431); and 52D4 (SEQ ID No. 432). The trispecific protein was purified into conditioned media from a stable clone pool using protein A and ion exchange chromatography. Fig.17 SDS-PAGE images of purified proteins are provided.

[0418] Affinity measurements against human and cynomolgus DLL3 were performed using 60 nM, 20 nM, 6.67 nM and 2.22 nM concentrations of biotinylated DLL3 targeting trispecific proteins immobilized on Octet streptavidin tips. The affinities determined from the measurements are listed in Table 10. In this experiment, the anti-DLL3 trispecific antibody containing DH6 (the parental DLL3 binder sequence of the affinity matured DLL3 binder sequence) had a K of 1.0 for human DLL3. D The value is 13.5 nM, and the K of cynomolgus monkey DLL3 DIn comparison, the K values ​​of the ten anti-DLL3 trispecific proteins, including affinity-matured DLL3 binder molecules tested in this experiment, were 11 nM. D The values ​​ranged from 0.9 to 2.2 nM for human DLL3 and from 1.4 to 3.4 nM for cynomolgus DLL3. Thus, the improvement in affinity ranged from 6.1 to 15-fold for human DLL3 and from 3.2 to 7.9-fold for cynomolgus DLL3.

[0419] The purified protein was tested in the TDCC assay using the same method as described in Example 2, except that two additional DLL3-expressing cell lines, DMS-53 and NCI-H510A, were included in the assay. The EC values ​​for these TDCC assays were 50 The values ​​are listed in Table 11, and the graphical representations of the DMS-53 and DMS-153 TDCC data are given in Figure 18-19 In these assays, trispecific molecules targeting GFP were inactive (e.g. Figure 18-19 Compared with the parental molecule DH6, the EC 50 The values ​​were improved by 2.3 to 12.1 times, and the EC 50 The values ​​were improved by 4.5 to 31.5 times, and the EC 50 The values ​​improved by 8.1 to 26.1 times.

[0420] Table 10: In vitro affinity of purified CHO-expressed affinity-matured anti-DLL3 trispecific proteins for human and macaque DLL3 proteins

[0421]

[0422]

[0423] Table 11: TDCC activity of purified CHO expressed affinity matured anti-DLL3 trispecific proteins using DMS153, NCI-H510A and DMS53 cell lines and human T cells

[0424]

[0425] Example 7: T cell targeting using an exemplary DLL3-targeting trispecific protein comprising a DLL3-binding protein of the present disclosure Cytotoxicity assay

[0426] Several exemplary DLL3 trispecific proteins comprising the DLL3 binding domain 52D04 (SEQ ID NO. 432) of the present disclosure 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 were shown in Table 1. Figure 22-24 In. Fig. 20 As shown, the trispecific protein Fig.21 As shown in the anti-DLL3: anti-ALB: anti-CD3 configuration (TAC) or as Fig.21 The anti-CD3: anti-ALB: anti-DLL3 (CAT) configuration includes a DLL3 binding domain, an albumin binding domain (anti-ALB), and a CD3 binding domain (anti-CD3). 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 titrated exemplary DLL3 binding trispecific proteins. It was hypothesized that if the DLL3 binding trispecific proteins directed T cells to kill NCI-H2171, DMS-79, SHP77, or WM2664 cells expressing DLL3, then the viability of these cells as determined by luciferase assays performed 48 hours after the start of the experiment should be reduced. Fig. 22 Shown are graphs of representative TDCC data for DLL3 binding tri-specific proteins containing the following DLL3 binding domains in either TAC or CAT configuration using NCI-H2171 cells. Fig.23 Shown are graphs of representative TDCC data using DMS-79 cells for DLL3 binding tri-specific proteins following DLL3 binding domains in either TAC or CAT configurations. Fig.24 Shown are graphs of representative TDCC data for DLL3 binding tri-specific proteins containing the following DLL3 binding domains in either TAC or CAT configuration using SHP77 cells. Fig.25 Graphs showing representative TDCC data for DLL3 binding trispecific proteins with the following DLL3 binding domains in TAC or CAT configurations using WM2664 cells. Table 12 lists the EC values ​​of the TDCC assay. 50 As shown in the figure and by EC 50The values ​​indicate that in the presence of human serum albumin (HSA), DLL3 with CAT orientation binds to the trispecific protein ( Fig.21 ) binds trispecific proteins more efficiently than DLL3 with the TAC configuration.

[0427] Table 12: TDCC activity of exemplary anti-DLL3 trispecific proteins using NCI-H2171, DMS-79, SHP77 and cell lines and human T cells

[0428]

[0429]

[0430] Embodiment 8: Binding of an exemplary DLL3-targeting trispecific protein to human T cells

[0431] In the cell binding study, human T cells were incubated in the presence or absence of exemplary DLL3 targeting trispecific proteins (in anti-DLL3: anti-ALB: anti-CD3 (TAC) configuration (SEQ ID No. 1891); or anti-CD3: anti-ALB: anti-DLL3 (CAT) configuration (SEQ ID No. 1890)). Human T cells were further incubated with a secondary antibody (anti-trispecific antibody) conjugated to Alexa Fluor 647 that recognizes the anti-albumin domain in the exemplary trispecific molecule. Binding of the anti-trispecific antibody was measured by flow cytometry. The binding of the anti-trispecific antibody was compared to cells incubated with the secondary antibody alone or without the exemplary trispecific protein or the secondary antibody ( Fig.26 Strong binding of the anti-trispecific antibody was observed in the presence of an exemplary DLL3 trispecific protein in the anti-DLL3: anti-ALB: anti-CD3 (TAC) configuration ( Fig.26 Compared to cells incubated with the secondary antibody alone or without incubation with the exemplary trispecific protein or the secondary antibody ( Fig. 27 Strong binding of the anti-trispecific antibody was also observed in the presence of an exemplary DLL3 trispecific protein in the anti-CD3: anti-ALB: anti-DLL3 (CAT) configuration ( Fig. 27 right peak in the middle graph).

[0432] Example 9: Binding of Exemplary DLL3-Targeting Trispecific Proteins to Cancer Cell Lines Expressing DLL3

[0433] In another binding study, cancer cells expressing DLL3 [NCI-H82 (lung cancer cell line), SHP77 (lung cancer cell line), DMS53 (lung cancer), or NCI-H2171 (lung cancer cell line)] were incubated with an exemplary DLL3-targeting trispecific molecule (in CAT or TAC configuration; 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 incubated with a secondary antibody conjugated to Alexa Fluor 647 or FITC that recognizes the anti-albumin domain in the trispecific molecule. Binding of the exemplary DLL3-targeting trispecific molecule or the control trispecific molecule to the cells was measured by flow cytometry. The binding of the exemplary DLL3-targeting trispecific molecule or the control trispecific molecule to the cells was significantly higher than that of the cells incubated with the control trispecific molecule targeting GFP ( Fig.28 The left peak in the middle graph) and strong binding of the DLL3-targeting trispecific protein (in TAC configuration) to each cell line was observed ( Fig.28 Compared to cells incubated with a control trispecific molecule targeting GFP ( Fig.29 The left peak in the middle graph) and strong binding of the DLL3 targeting trispecific protein (in the CAT configuration) to each cell line was observed ( Fig.28 In control experiments using cell lines HCTI16 (colon cancer cell line) and NCI-H292 (lung cancer cell line) that lack DLL3 expression, similar amounts of anti-trispecific antibodies bound to cells incubated with the exemplary DLL3-targeting trispecific protein or a control trispecific molecule targeting GFP (data not shown), indicating that the exemplary DLL3-targeting trispecific molecule did not bind to cells that lack DLL3 expression.

[0434] Example 10: Exemplary DLL3-targeting trispecific proteins direct T cell-mediated targeting of DLL3-expressing cancer cells The killing ability of the system

[0435] The aim of this study was to evaluate whether exemplary DLL3-targeting trispecific molecules could direct T cells to kill DLL3-expressing cell lines NCI-H82, SHP77, DMS53, and NCI-H2171. The DLL3-expressing cells used in this study were engineered to express luciferase.

[0436] For the TDCC assay (T cell-dependent cytotoxicity assay), T cells from four healthy donors (Donor 2; Donor 47; Donor 81; Donor 86) were mixed with cells expressing DLL3, and different amounts of exemplary DLL3-targeting trispecific proteins (in CAT or TAC configuration; SEQ ID No. 1890 and SEQ ID No. 1891) were added to the mixture. The mixture was incubated at 37°C for 48 hours. As a control, a parallel experiment was performed using a control trispecific molecule targeting GFP. After 48 hours, the remaining live cells expressing DLL3 were quantified using a luminescence assay. It was observed that the DLL3-targeting trispecific molecule (in both TAC and CAT configurations) was able to effectively direct T cells from all four healthy donors to kill all four DLL3-expressing cell lines (for results using the TAC configuration, see Fig.30 , 31 , 32 and 33; results using the CAT configuration are shown in Fig.34 , 35 , 36 and 37), whereas the control GFP TriTAC molecule was unable to do so (also shown in Figure 30-37 EC 50 The values ​​are shown in Table 13 and Table 14. Further TDCC assays were performed using TriTAC targeting DLL3 and cell lines NCI-H292 and HCT116 lacking DLL3 expression. It was observed that TriTAC targeting DLL3 could not direct T cells to kill these two cell lines lacking DLL3 expression (data not shown).

[0437] Table 13: EC of TDCC assay using exemplary DLL3 targeting trispecific proteins comprising the DLL3 binding domain 52D04 of the present disclosure in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. 50 value.

[0438]

[0439] Table 14: EC of TDCC assay using exemplary DLL3 targeting trispecific protein comprising the DLL3 binding domain 52D04 of the present disclosure in the anti-CD3: anti-ALB: anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA) using T cells from four different donors. 50 value.

[0440]

[0441]

[0442] Example 11: DLL3-dependent activation of T cells by an exemplary DLL3-targeting trispecific protein

[0443] In this assay, T cells from 4 different healthy donors (Donor 2; Donor 35; Donor 47; and Donor 86) and NCI-H82 or DMS53 cells were incubated with DLL3 targeting trispecific proteins (in CAT or TAC configuration; SEQ ID No. 1890 and SEQ ID No. 1891) at 37°C for 48 hours. T cells from the same donors were also incubated with a control trispecific molecule GFP TriTAC targeting GFP and NCI-H82 or DMS53 cells at 37°C for 48 hours. After 48 hours, T cells were collected and CD69 and CD25 expression on T cells was measured by flow cytometry. Figure 38-45 As shown, increased CD69 or CD25 expression was detected on T cells from all 4 healthy donors in the presence of NCI-H82 or SHP77 cells and DLL3-targeting trispecific molecules, but not in the presence of the negative control GFP TriTAC. Parallel experiments were performed using HCT116 cells that lack DLL3 expression. No increase in CD69 or CD25 expression was observed with the DLL3 trispecific molecules tested in HCT116 cells (data not shown).

[0444] Example 12: DLL3-dependent cells generated by T cells induced by exemplary DLL3-targeting trispecific proteins factor

[0445] In this assay, T cells from healthy donors and NCI-H82 or SHP77 cells were incubated with exemplary DLL3-targeting trispecific molecules (in CAT or TAC configuration; SEQ ID No.1890 and SEQ ID No.1891) at 37°C for 48 hours. T cells from the same donor were also incubated with a control trispecific molecule targeting GFP, GFP TriTAC, and NCI-H82 or DMS53 cells at 37°C for 48 hours. After 48 hours, the conditioned medium was collected and the amount of various cytokines present in the conditioned medium was measured using an electrochemiluminescence assay (Meso Scale Discovery). It was observed that IFNγ, IL-2, and TNFα were secreted into the culture medium in the presence of NCI-H82 or SHP77 cells and the DLL3-targeting trispecific molecules, but not in the presence of the control TriTAC molecules targeting GFP. For the DLL3-targeting trispecific molecules in the TAC configuration: IFNγ production was Fig.46 and 47 IL-2 production in Fig.48 and 49 The production of TNFα is shown in Fig.50 and 51For the DLL3 targeting trispecific molecule in CAT configuration: IFNγ production in Fig.52 and 53 IL-2 production in Fig.54 and 55 The production of TNFα is shown in Fig.56 and 57 Shown in.

[0446] Example 13: Inhibition of NCI-H82 xenograft growth by an exemplary DLL3-targeting trispecific protein

[0447] For this study, 5x 10 6 Human T cells and 5x 10 6 NCI-H82 small cell lung cancer cells were injected into mice. On days 1 to 10, mice were injected intraperitoneally (ip) daily with 20, 100 or 500 μg / kg doses of an exemplary DLL3-targeted trispecific molecule (in CAT or TAC configuration; SEQ ID No. 1890 and SEQ ID No. 1891), or a negative control TriTAC targeting GFP at a dose of 500 μg / kg. Tumor volume was measured every few days starting from day 7 and ending on day 24. Significant inhibition of tumor growth was observed in mice injected with the DLL3-targeted trispecific protein at all doses, as compared to mice given the TriTAC targeting GFP at a dose of 500 μg / kg. Fig.58 shown.

[0448] Example 14: Exemplary DLL3-targeted trispecific protein eliminates NCI-H82 xenografts

[0449] For this study, 5x10 6 NCI-H82 small cell lung cancer cells. Mice were randomized on day 8 and each mouse was injected with 2x10 7 On days 9 to 18, mice were injected intraperitoneally daily with 1, 10, or 100 μg / kg doses of an exemplary DLL3-targeting trispecific molecule (in CAT configuration; SEQ ID No. 1890) or 100 μg / kg doses of a negative control TriTAC targeting GFP. Tumor volume was measured every few days starting on day 8 and ending on day 29. Significant inhibition of tumor growth was observed in mice injected with the DLL3-targeting trispecific molecule at doses of 10 and 100 μg / kg, as compared to mice given the TriTAC targeting GFP at a dose of 100 μg / kg. Fig.59 shown.

[0450] Example 15: Inhibition of SHP77 xenograft growth by an exemplary DLL3-targeting trispecific protein

[0451] For this study, on day 0, 5x10 6Human T cells and 5x10 6 SHP77 small cell lung cancer cells were injected into mice. On days 1 to 10, mice were injected intraperitoneally with 1, 10 or 100 μg / kg doses of DLL3-targeted trispecific molecules (in CAT configuration; SEQ ID No. 1890), or 100 μg / kg doses of a negative control TriTAC targeting GFP. Tumor volume was measured every few days starting from day 6 and ending on day 28. Significant inhibition of tumor growth was observed in mice injected with DLL3-targeted trispecific proteins at doses of 10 and 100 μg / kg, as compared to mice given TriTAC targeting GFP at a dose of 100 μg / kg. Fig.60 shown.

[0452] Example 16: Pharmacokinetic profiles of exemplary DLL3-targeted trispecific proteins

[0453] The half-life of the DLL3-targeted trispecific protein in cynomolgus monkeys was about 3 days to about 3.9 days when dosed at 0.3 mg / kg

[0454] For this study, cynomolgus monkeys were injected intravenously with a 0.3 mg / kg dose of an exemplary DLL3-targeting trispecific molecule (in CAT or TAC configuration; SEQ ID No. 1890 and SEQ ID No. 1891), and serum samples were collected at different time points after injection. Two monkeys were injected per dose. The amount of DLL3-targeting trispecific molecule in serum was measured in an electrochemiluminescent assay using an anti-idiotypic antibody that recognizes the trispecific molecule. Fig.61 A graph showing serum levels of the DLL3 targeting trispecific molecule at different time points is shown. The data were then used to calculate the pharmacokinetic properties of the DLL3 targeting trispecific molecule, as shown in Table 15. Based on the pharmacokinetic data, a once or twice weekly dosing schedule for humans can be considered.

[0455] Table 15: Pharmacokinetics of exemplary DLL3-targeting trispecific molecules

[0456]

[0457] The half-life of the DLL3-targeted trispecific protein in cynomolgus monkeys was about 2.8 to about 3.3 days when dosed at 1 or 10 mg / kg:

[0458] For this study, cynomolgus monkeys were injected intravenously with an exemplary DLL3-targeting trispecific molecule at a dose of 1 mg / kg or 10 mg / kg, and serum samples were collected at different time points after injection. Two monkeys were injected per dose. The amount of DLL3-targeting TriTAC in the serum was measured in an electrochemiluminescent assay using an anti-idiotypic antibody that recognizes the TriTAC molecule. Fig.62 A graph showing serum levels of the DLL3 targeting trispecific molecule at different time points is shown. The data were then used to calculate the pharmacokinetic properties of this TriTAC molecule as shown in Table 16. The pharmacokinetic data suggest once or twice weekly dosing.

[0459] Table 16: Pharmacokinetics of exemplary DLL3-targeting trispecific molecules

[0460]

[0461] Exemplary DLL3-targeting trispecific proteins were tolerated in cynomolgus monkeys when given at a single dose of up to 10 mg / kg:

[0462] A transient increase in serum cytokine levels was observed, primarily when the exemplary DLL3-targeting trispecific protein (in CAT configuration) was administered at a dose of 10 mg / kg ( Fig.63 IFNγ- Fig.63 Above, IL-6 Fig.63 Second picture: IL-10 Fig.63 Transient T cell marginalization and T cell activation were also observed (data not shown). No macroscopic findings or differences in organ weights associated with the DLL3 trispecific protein were observed at terminal and recovery euthanasia, while no microscopic findings associated with the DLL3 trispecific protein were observed at recovery euthanasia.

[0463] To demonstrate that the DLL3-targeted TriTAC retained cell-directed killing activity following administration to cynomolgus monkeys, serum samples from the 10 mg / kg dose group collected 168 hours after dosing were tested in the DMS53 TDCC assay and compared to freshly thawed DLL3-targeted TriTAC. Equivalent cell DMS53 cell killing was observed for serum samples and freshly thawed protein ( Fig.64 ), indicating that DLL3-targeting TriTAC retained the ability to guide T cells to kill target cells after 1 week of administration in cynomolgus monkeys.

[0464] Example 17: Xenograft Tumor Model

[0465] Exemplary anti-DLL3 targeting trispecific proteins of the present disclosure were evaluated in a xenograft model.

[0466] Female immunodeficient NOD / scid mice were sublethally irradiated (2 Gy) and 1x10 6 NCI-H28 cells were inoculated subcutaneously into their right dorsal flank. When tumors reached 100 to 200 mm 3 The animals were divided into three treatment groups. Groups 2 and 3 (8 animals each) were injected intraperitoneally with 1.5x107 Three days later, animals in Group 3 were treated with a total of 9 intravenous doses (e.g., 1, 10, 50, or 100 μg / kg) of an exemplary DLL3 trispecific antigen binding protein (qd x 9d). Groups 1 and 2 were treated with vehicle only. Body weight and tumor volume were measured for 30 days.

[0467] Animals treated with the exemplary DLL3-targeting tri-specific proteins of the foregoing Examples are expected to have a statistically significant delay in tumor growth compared to corresponding vehicle-treated controls.

[0468] Example 18: Administration of an exemplary DLL3 trispecific antigen binding protein (anti-DLL3 trispecific protein) Proof-of-Concept Clinical Trial Protocol for Patients with Neuroendocrine Cancers

[0469] This is a Phase I / II clinical trial investigating an exemplary DLL3 trispecific antigen binding protein as a treatment for neuroendocrine cancers.

[0470] Study outcome measures:

[0471] Primary: Maximum tolerated dose of an exemplary DLL3-targeting trispecific protein.

[0472] Secondary: Determine whether in vitro responses of the exemplary DLL3-targeting trispecific protein correlate with clinical responses

[0473] Phase I

[0474] The maximum tolerated dose (MTD) was determined in the Phase I portion of the trial.

[0475] 1.1 The maximum tolerated dose (MTD) was determined in the Phase I part of this trial.

[0476] 1.2 Patients meeting the eligibility criteria are enrolled in the trial to evaluate the exemplary DLL3-targeting trispecific protein.

[0477] 1.3 The goal is to determine the highest dose of an exemplary anti-DLL3 trispecific protein that can be safely administered in a participant without severe or difficult to manage side effects. The dose given depends on the number of participants who have been previously enrolled in the study and the dose tolerance. Not all participants receive the same dose.

[0478] Phase II

[0479] 2.1 The subsequent Phase II portion was treated at the MTD with the goal of determining whether therapy with the exemplary DLL3-targeting trispecific protein would result in a response rate of at least 20%.

[0480] The primary outcome of Phase II is to determine whether therapy with the exemplary DLL3-targeted trispecific protein will result in a clinical response (blast response, minor response, partial response, or complete response) in at least 20% of patients.

[0481] Eligibility: Biopsy-confirmed neuroendocrine tumors with somatostatin receptor positivity confirmed by somatostatin receptor PET.

[0482] All locations or sources are eligible.

[0483] Functioning and non-functioning tumors were allowed.

[0484] Not suitable for surgical reduction.

[0485] ECOG performance status of 0, 1, or 2

[0486] Age > 18 years old.

[0487] Be able to understand the written informed consent document and be willing to sign it.

[0488] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will now appreciate that many variations, changes and substitutions may be employed in the process of implementing the present invention. It is intended that the scope of the present invention be limited to the appended claims, thereby encompassing methods and structures and their equivalents within the scope of these claims.

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[0550] Connector

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[0553] CD3 binding domain CDR sequences

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[0558] DLL3 Protein UniProtKB Accession Q9NYJ7 (SEQ ID NO: 1885)

[0559] >sp|Q9NYJ7|DLL3_Human Delta-like protein 3 OS=Homo sapiens OX=9606 GN=DLL3 PE=1 SV=1 MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYLLPPALGLLVAAGVAGAALLLVHVRRRGHSQDAGSRLLAGTPEPSVHALPDALNNLRTQEGSGDGPSSSVDWNRPEDVDPQGIYVISAPSIYAREVATPLFPPLHTGRAGQRQHLLFPYPSSILSVK

[0560] 51X5(SEQ ID NO:1886)

[0561] EVQLVESGGGLVQPGGSLTLSCAASLSSVSVLSIAWYRQAPGKKRELVAGISTDGSTVYIDSVKGRFTISRD

[0562] NAKNSVYLQMNSLRAEDTAVYYCYAYSWTTSLPYWGQGTLVTVSS

[0563] 51X5 CDR1(SEQ ID NO:1887)

[0564] LSSVSVLSIA

[0565] 51X5 CDR2 (SEQ ID NO:1888)

[0566] GISTDGSTVYIDSVKG

[0567] 51X5 CDR3 (SEQ ID NO:1889)

[0568] YSWTTSLPY

[0569] >NP_058637.1 delta-like protein 3 isoform 1 precursor [Homo sapiens] (SEQ ID No.1892)

[0570] MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALG

[0571] FGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQR

[0572] YLLPPALGLLVAAGVAGAALLLVHVRRRGHSQDAGSRLLAGTPEPSVHALPDALNNLRTQEGSGDGPSSSVDWNRPEDVDPQGIYVISAPSIYAREVATPLFPPLHTGRAGQRQHLLFPYPSSILSVK

[0573] DLL3 protein sequence (SEQ ID NO:1893)

[0574] RSPCSARLPCRLFFRVCLKPGLSEEAAESPCALGAALSARGPVYTEQPGAPAPDLPLPDGLLQVPFRDAWPGTFSFIIETWREELGDQIGGPAWSLLARVAGRRRLAAGGPWARDIQRAGAWELRFSYRARCEPPAVGTACTRLCRPRSAPSRCGPGLRPCAPLEDECEAPLVCRAGCSPEHGFCEQPGECRCLEGWTGPLCTVPVSTSSCLSPRGPSSATTGCLVPGPGPCDGNPCANGGSCSETPRSFECTCPRGFYGLRCEVSGVTCADGPCFNGGLCVGGADPDSAYICHCPPGFQGSNCEKRVDRCSLQPCRNGGLCLDLGHALRCRCRAGFAGPRCEHDLDDCAGRACANGGTCVEGGGAHRCSCALGFGGRDCRERADPCAARPCAHGGRCYAHFSGLVCACAPGYMGARCEFPVHPDGASALPAAPPGLRPGDPQRYL

Claims

1. A DLL3-targeting trispecific protein, wherein the protein comprises (I) a first domain (A) that can specifically bind to human CD3, which is a single-chain variable fragment (scFv); (II) a second domain (B) which is a single domain antibody (sdAb) that specifically binds to human serum albumin; and (III) a third domain (C) capable of specifically binding to a DLL3 protein, which is a single domain antibody (sdAb), wherein the third domain (C) comprises: i) CDR1 consisting of the amino acid sequence of SEQ ID NO: 874, CDR2 consisting of the amino acid sequence of SEQ ID NO: 1316, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 1758; ii) CDR1 consisting of the amino acid sequence of SEQ ID NO: 851, CDR2 consisting of the amino acid sequence of SEQ ID NO: 1293, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 1735; iii) CDR1 consisting of the amino acid sequence of SEQ ID NO: 867, CDR2 consisting of the amino acid sequence of SEQ ID NO: 1309, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 1751; iv) a CDR1 consisting of the amino acid sequence of SEQ ID NO: 872, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 1314, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 1756; v) CDR1 consisting of the amino acid sequence of SEQ ID NO: 873, CDR2 consisting of the amino acid sequence of SEQ ID NO: 1315, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 1757; or vi) CDR1 consisting of the amino acid sequence of SEQ ID NO: 1887, CDR2 consisting of the amino acid sequence of SEQ ID NO: 1888, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 1889; wherein the third domain (C) comprises an amino acid sequence having at least about 85%-95% sequence identity with SEQ ID NO:75; wherein the domains are connected in the order of H2N-(A)-(B)-(C)-COOH or H2N-(C)-(B)-(A)-COOH.

2. The DLL3 targeting tri-specific protein according to claim 1, wherein the third domain (C) comprises the sequence of SEQ ID No.

408.

3. The DLL3 targeting tri-specific protein according to claim 1, wherein the third domain (C) comprises the sequence of SEQ ID No.

425.

4. The DLL3 targeting tri-specific protein according to claim 1, wherein the third domain (C) comprises the sequence of SEQ ID No.

430.

5. The DLL3 targeting tri-specific protein of claim 1, wherein the third domain (C) comprises the sequence of SEQ ID NO:

431.

6. The DLL3 targeting tri-specific protein of claim 1, wherein the third domain (C) comprises the sequence of SEQ ID NO:

432.

7. The DLL3 targeting tri-specific protein of claim 1, wherein the third domain (C) comprises the sequence of SEQ ID NO: 1886.

8. The DLL3 targeting tri-specific protein according to claim 1, wherein the second domain (B) comprises a sequence selected from SEQ ID No. 1769-1778.

9. The DLL3 targeting tri-specific protein according to claim 1, wherein the second domain (B) comprises the sequence of SEQ ID No. 1890.

10. The DLL3 targeting tri-specific protein of claim 1, wherein the second domain (B) comprises the sequence of SEQ ID NO: 1891.

11. The DLL3 targeting tri-specific protein according to claim 1, wherein the first domain (A) comprises a sequence selected from the group consisting of SEQ ID Nos. 1793-1807.

12. The DLL3 targeting tri-specific protein of claim 1, wherein domains C and B are connected by a linker L1, and domains B and A are connected by a linker L2.

13. The DLL3 targeting trispecific protein according to claim 12, wherein linkers L1 and L2 are each independently selected from (GS) n (SEQ ID No.1809), (GGS) n (SEQ ID No. 1810), (GGGS) n (SEQ ID No.1811), (GGSG) n (SEQ ID No.1812), (GGSGG) n (SEQ ID No.1813) or (GGGGS) n (SEQ ID No. 1814), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or GGGGSGGGS (SEQ ID No. 1808).

14. The DLL3 targeting trispecific protein according to claim 13, wherein linkers L1 and L2 are each independently (GGGGS)4 (SEQ ID No. 1817), (GGGGS)3 (SEQ ID No. 1818) or GGGGSGGGS (SEQ ID No. 1808).

15. The DLL3 targeting tri-specific protein of claim 1, wherein the protein is less than about 80 kDa.

16. The DLL3 targeting tri-specific protein of claim 1, wherein the protein has an elimination half-life of at least about 50 hours.

17. The DLL3 targeting tri-specific protein of claim 1, wherein the domains are connected in the order of H2N-(A)-(B)-(C)-COOH.

18. Use of a DLL3 targeting trispecific protein according to any one of claims 1 to 17 in the preparation of a medicament for treating a cancer expressing DLL3.

19. The use according to claim 18, wherein the cancer expressing DLL3 comprises lung cancer, gastric cancer, ovarian cancer, prostate cancer, neuroendocrine cancer, or triple-negative breast cancer.

20. The use according to claim 19, wherein the cancer expressing DLL3 comprises lung cancer, and the lung cancer is small cell lung cancer.

21. A polynucleotide encoding a DLL3 targeting tri-specific protein according to any one of claims 1-17.

22. A vector comprising the polynucleotide according to claim 21.

23. A host cell comprising the polynucleotide according to claim 21.

24. A host cell comprising the vector of claim 22.

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