TRISPECIFIC PROTEIN TARGETING DLL3, POLYNUCLEOTIDE, VECTOR, HOST CELL, ITS USES, METHOD AND PROCESS FOR PRODUCING IT, AND PHARMACEUTICAL COMPOSITION

Description

"TRISPECIFIC PROTEIN TARGETING DLL3, POLYNUCLEOTIDE, VECTOR, HOST CELL, ITS USES, METHOD AND PROCESS FOR PRODUCING IT AND PHARMACEUTICAL COMPOSITION (Divided from BR 112021005769-2, filed on 09 / 25 / 2019) CROSS-REFERENCE"

[0001] This application claims the benefit of the Applications Provisional Applications No. US 62 / 736,368 filed on September 25, 2018; 62 / 736,358 filed on September 25, 2018; and 62 / 877.227 filed on July 22, 2019, each of which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] This application contains a sequence listing that was submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on September 25, 2019, is named 47517-733_601_SL.txt and is 951,519 bytes in size. BACKGROUND OF THE INVENTION

[0003] The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, a primary goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and tissues intact and undamaged. One such method is to induce an immune response against the tumor, causing immune effector cells, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs), to attack and destroy tumor cells. SUMMARY OF THE INVENTION

[0004] There is still a need for additional options to be available for the treatment of tumor diseases related to DLL3 overexpression, such as neuroendocrine tumors. A Petition 870260081775, dated 08 / 13 / 2026, page 10 / 38 2 / 256 The present disclosure provides, in certain embodiments, single-domain proteins that specifically bind to DLL3 on the surface of target tumor cells and multispecific proteins, such as trispecific proteins containing a DLL3-binding domain, as described in this document. In some embodiments, the present disclosure provides Delta-Like Ligand-binding 3 (DLL3) proteins, or multispecific proteins as mentioned above, that can be used to diagnose and treat indications correlated to DLL3 expression.

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

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

[0007] In some embodiments, the third domain (C) comprises an affinity-matured binding molecule. In some embodiments, the affinity-matured binding molecule is derived from a parent molecule that specifically binds to the DLL3 protein. In some embodiments, the affinity-matured binding molecule has a binding affinity for the DLL3 protein that is about 2 times to about 50 times greater than the Petition 870260057806, dated 06 / 15 / 2026, p. 13 / 374 3 / 256 binding affinity of the parental molecule for the DLL3 protein. In some embodiments, the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886. In some embodiments, the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-52. In some embodiments, the third domain (C) comprises a CDR1, a CDR2, and a CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 443, 884, and 1887, wherein CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 885-1326 and 1888, and wherein CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 495-528.In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 937-970. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1379-1412. In some embodiments, the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID Nos. 53-86. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 529-809. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 971 to 1251. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1379-1412. In some forms, the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID Nos. 87367.In some modalities, CDR1 comprises a sequence. Petition 870260057806, dated 06 / 15 / 2026, p. 14 / 374 4 / 256 selected from the group consisting of SEQ ID Nos. 810884. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 1252 to 1326. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1692 to 1768. In some embodiments, the trispecific protein targeting DLL3, according to claim 1, wherein the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID Nos. 368-442.

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

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

[0010] In some modalities: X1 is A, D, E, F, G, H, K, L, M, N, Q, R, S, V, W or Y; X2 is D, E, H, K, M, P, R, S, T or Y; X3 is A, D, G, H, K, N, P, Q, R, S, T, V or Y; X4 is K, S, or V; X5 is A, F, G, H, K, L, M, N, Q, R, S, T, V, W or Y; Petition 870260057806, dated 06 / 15 / 2026, p. 15 / 374 5 / 256 X6 is D, F, H, I, K, L, M, N, Q, R, S, V or Y; X7 is L or M; and X8 is I, L, M, S, T, or V.

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

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

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

[0014] One embodiment provides a DLL3-binding protein comprising the following formula: f1-r1-f2-r2-f3-r3-f4 Petition 870260057806, dated 06 / 15 / 2026, p. 16 / 374 6 / 256 where, 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 where f1, f2, f3 and f4 are structural residues.

[0015] One embodiment provides a method for the treatment or improvement of a proliferative disease, or a tumorous disease, comprising the administration of the trispecific protein targeting DLL3, according to this disclosure, to an individual in need thereof.

[0016] One embodiment provides a trispecific protein that targets DLL3, wherein said protein comprises (a) a first domain (A) that specifically binds to human CD3; (b) a second domain (B) that is a half-life extension domain; and (c) a third domain (C) that specifically binds to a DLL3 protein. In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, H2N-(C)-(B)-(A)-COOH, or H2N-(C)-(A)-(B)-COOH. In some embodiments, the third domain (C) comprises an affinity-matured binding molecule. In some embodiments, the affinity-matured binding molecule is derived from a parent molecule that specifically binds to the DLL3 protein. In some embodiments, the affinity-matured binding molecule is derived from the parent molecule that specifically binds to DLL3, after an affinity maturation cycle.In some modalities, the affinity-based maturation cycle involves positioning a library. Petition 870260057806, dated 06 / 15 / 2026, p. 17 / 374 7 / 256 of phage display against DLL3 protein. In some embodiments, the phage display library is generated by mutation of 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 molecule is selected from one or more molecules derived from the parent molecule. In some embodiments, the affinity-matured DLL3-binding molecule has a higher binding affinity for the DLL3 protein than the binding affinity of the parent molecule for the DLL3 protein. In some embodiments, the affinity-matured binding DLL3-binding molecule has a binding affinity for the DLL3 protein that is about 2 times to about 50 times higher than the binding affinity of the parent molecule for the DLL3 protein.In some embodiments, the affinity-matured DLL3-binding molecule has a binding affinity for the DLL3 protein that is approximately 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 approximately 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 approximately 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-52. In some embodiments, the third domain (C) comprises a CDR1, a CDR2, and a CDR3.

[0017] In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 443-884 and 1887, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 443-884 and 1887. In some embodiments, CDR2 comprises a sequence selected from the group Petition 870260057806, dated 06 / 15 / 2026, p. 18 / 374 8 / 256 consisting of SEQ ID Nos. 885-1326 and 1888, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 885-1326 and 1888. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889, or one or more substitutions relative to a sequence selected from SEQ ID Nos. 1327-1768 and 1889. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 495-528, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 495-528. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 937-970, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 937-970.In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1379-1412, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 1379 to 1412.

[0018] In some embodiments, the third domain (C) comprises a sequence that is at least about 80% identical to a sequence selected from the group consisting of SEQ ID Nos. 53-86. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 529-809, or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 529-809. In some embodiments, CDR2 comprises a sequence having one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 971 to 1251. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1379-1412, or one or more substitutions Petition 870260057806, dated 06 / 15 / 2026, p. 19 / 374 9 / 256 of amino acids relative to a sequence selected from the group consisting of SEQ ID Nos. 1379 to 1412. In some embodiments, the third domain (C) comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 87-367. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 810-884, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 810 to 884. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 1252 to 1326, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 1252 to 1326. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos.1692 to 1768, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 1692 to 1768. In some embodiments, the third domain (C) comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 368-442.

[0019] One embodiment provides a trispecific protein targeting DLL3, wherein said protein comprises (a) a first domain (A) that specifically binds to human CD3; (b) a second domain (B) that is a half-life extension domain; and (c) a third domain (C) that specifically binds to DLL3, wherein the third domain comprises the sequence 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 a CDR1, a CDR2, and a CDR3. In some embodiments, CDR1 comprises the following sequence: GX1X2X3X4X5NX6X7X8. In some embodiments, CDR2 comprises the following sequence: GJ1SJ2J3J4J5J6J7J8J9J10SJ11KJ12 (SEQ ID Petition 870260057806, dated 06 / 15 / 2026, p. 20 / 374 10 / 256 No. 1895).

[0020] In some forms, CDR3 comprises the following sequence: Z1Z2Z3Z4Z5Z6Z7Z8Z9Z10Z11. In some forms: X1 is A, E, F, G, I, K, L, N, Q, R, S, T, V or Y; X2 is A, G, I, K, P, R, S, T, or V; X3 is A, D, F, K, L, N, P, Q, R, S, T or Y; X4 is A, D, F, H, I, K, L, M, N, P, R, S, T, V or Y; X5 is F, I, K, L, M, N, R, S, T or V; X6 is either A or G; X7 is F, I, L, M, T, V or Y; and X8 is either A or G.

[0021] In some modalities: J1 is either I or V; J2 is A, K, P, R, or S; J3 is either D or N; J4 is D, E, G, K, N, Q, R, S, T or Y; J5 is either S or T; J6 is A, E, F, H, I, K, L, N, Q, R, S, T, V or Y; J7 is A, I, L, M, V, or Y; J8 is D, F, H, I, L, N, S, T, V or Y; J9 is A, D, E, F, G, I, K, L, N, Q, R, S, T, V or Y; J10 is A, D, E, G, K, Q, S, or V; J11 is either A or V; and J12 is G or V.

[0022] In some modalities: Z1 is either F or Y; Z2 is G, H, I, K, N, R, S, or T; Z3 is A, F, H, I, K, L, M, N, P, Q, R, S, T or Y; Z4 is A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V or Y; Z5 is A, C, D, E, G, H, I, K, L, N, P, Q, R, S, T, W or Y; Z6 is G, K, L, R, or T; Z7 is A, G, H, L, Q, R, S, T, V or Y; Z8 is A, D, E, G, H, P, Q, S, T, W or Y; Petition 870260057806, dated 06 / 15 / 2026, p. 21 / 374 11 / 256 Z9 is A, G, I, K, L, M, N, Q, R, S, T, V, or Y; Z10 is A, G, K, P, R, S, T or V; and Z11 is A, F, S or Y.

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

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

[0025] In some modalities: J1 is either I or V; J2 is A, D, E, G, H, I, K, L, N, P, Q, R, S, T, V, or Y; J3 is A, D, E, G, H, N, R, or T; J4 is H, P, R, or S; J5 is A, H, I, K, M, N, Q, R, T or V; J6 is A, D, G, H, I, L, M, N, S, T, Vou Y; J7 is A, F, I, L, M, R, S, T, V or Y; J8 is A, D, E, G, H, K, L, N, R, S or V. Petition 870260057806, dated 06 / 15 / 2026, p. 22 / 374 12 / 256

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

[0027] In some embodiments, the third domain (C) comprises a humanized antibody or an antigen-binding fragment thereof. In some embodiments, the third domain (C) comprises a single-domain antibody, a VHH domain, an 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 serum protein in bulk. In some embodiments, the second domain (B) comprises a single-domain antibody, a VHH domain, an 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 serum protein in bulk.In some embodiments, the second domain (B) comprises the single-domain antibody that specifically binds to the serum protein in volume. In some embodiments, the serum protein in volume comprises albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, Fibrinogen, IgE, pentameric IgM, or free IgK light chain. In some embodiments, the serum protein in volume comprises albumin. In some embodiments, the second domain (B) comprises a sequence that is at least about 75%. Petition 870260057806, dated 06 / 15 / 2026, page 23 / 374 13 / 256 identical to a sequence selected from the group consisting of SEQ ID Nos. 1769-1778. In some embodiments, the second domain (B) comprises a sequence that is at least about 75% identical to SEQ ID No. 1774.

[0028] In some embodiments, the first domain (A) comprises a single-domain antibody, a VHH domain, an 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 the group consisting of SEQ ID Nos. 17931807. In some embodiments, the third domain (C) comprises the following formula: f1-r1-f2-r2-f3-r3-f4 where, r1 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. 443884 and 1887; r2 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. 8851326 and 1888; and r3 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. 13271768 and 1889; and where f1, f2, f3 and f4 are structural residues. In some embodiments, the third domain (C) comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886. In some embodiments, domains C and B are connected by L1 linker and domains B and A are connected by L2 linker.In some embodiments, the L1 and L2 ligands are each selected independently 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. Petition 870260057806, dated 06 / 15 / 2026, p. 24 / 374 14 / 256 (SEQ ID No. 1814), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the L1 and L2 ligands 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 DLL3, or both human and cynomolgus DLL3. In some embodiments, the protein is smaller than about 80 kDa. In some embodiments, the protein is from about 50 to about 75 kDa. In some embodiments, the protein is smaller than about 60 kDa. In some forms, the protein has a half-life of at least about 50 hours. In some forms, the protein has a half-life of at least about 100 hours. In some forms, the protein has increased tissue penetration compared to an IgG for the same DLL3.In some embodiments, the domains are linked in the order H2N(A)-(B)-(C)-COOH.

[0029] One embodiment provides a pharmaceutical composition comprising (i) the DLL3-targeting trispecific protein according to any of the above embodiments, or a DLL3-binding protein according to such disclosure, and (ii) a pharmaceutically acceptable carrier. One embodiment provides a method for preparing a DLL3-targeting trispecific binding protein according to any of the above embodiments, wherein the method comprises: (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 from the library a DLL3-binding protein that specifically binds to said oligomer or derivative thereof; and (iv) preparing the 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 protein. Petition 870260057806, dated 06 / 15 / 2026, p. 25 / 374 15 / 256 of DLL3 in vitro by screening the recombinant library with said DLL3 protein. In some embodiments, the recombinant library is expressed on the surface of a bacteriophage. In some embodiments, the recombinant library is expressed on the surface of yeast cells. In some embodiments, the recombinant library is expressed on the surface of bacterial cells. In some embodiments, the recombinant library is expressed on RNA-protein fusions. In some embodiments, the recombinant library is an scFv library or a Fab library. In some embodiments, the recombinant antibody library is a single-domain library.

[0030] One embodiment provides a process for the production of a trispecific protein targeting DLL3, according to any of the embodiments above, wherein said process comprises cultivating a transformed or transfected host with a vector comprising a nucleic acid sequence encoding the trispecific protein DLL3, according to any of the embodiments above, under conditions that permit the expression of the trispecific protein targeting DLL3 and recovering and purifying the protein produced from the culture.

[0031] One embodiment provides a DLL3-binding protein comprising the following formula: f1-r1-f2-r2-f3-r3-f4 where, 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 Petition 870260057806, dated 06 / 15 / 2026, p. 26 / 374 16 / 256 1768 and 1889; and where f1, f2, f3, and f4 are structural residues. In some embodiments, the DLL3-binding protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886. In some embodiments, the DLL3-binding protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-52. In some embodiments, the DLL3-binding protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID 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 the group consisting of SEQ ID Nos. 87-367.In some embodiments, the DLL3-binding protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 368442. One embodiment provides the DLL3-binding protein that is derived from a parental DLL3-binding protein comprising the sequence of SEQ ID No. 68. Another embodiment provides the DLL3-binding protein that is derived from a parental DLL3-binding protein comprising the sequence of SEQ ID No. 75 or comprises the sequence of SEQ ID No. 75.

[0032] One embodiment provides a method for preparing DLL3-binding protein in accordance with this disclosure, and the method comprises: 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 from the library a DLL3-binding protein that specifically binds to said oligomer or derivative thereof. In some embodiments, the recombinant library of DLL3-binding proteins is exposed to the DLL3 protein in vitro. Petition 870260057806, dated 06 / 15 / 2026, p. 27 / 374 17 / 256 by screening the recombinant library with said DLL3 protein. In some embodiments, the recombinant library is expressed on the surface of a bacteriophage. In some embodiments, the recombinant library is expressed on the surface of yeast cells. In some embodiments, the recombinant library is expressed on the surface of bacterial cells. In some embodiments, the recombinant library is expressed on RNA-protein fusions. In some embodiments, the recombinant library is an scFv library or a Fab library. In some embodiments, the recombinant antibody library is a single-domain library.

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

[0034] An embodiment provides a method for the treatment or improvement of a proliferative disease, or a tumorous disease, comprising the administration of the DLL3-targeting trispecific protein, according to any of the embodiments above, a DLL3-binding protein, according to such disclosure, or a pharmaceutical composition as provided herein, to an individual in need thereof. In some embodiments, the individual is a human being. In some embodiments, the method further comprises the administration of an agent in combination with the DLL3-targeting trispecific protein, according to any of the embodiments above, wherein the DLL3-binding protein, according to such disclosure, or the pharmaceutical composition as provided herein. In some embodiments, the DLL3-targeting trispecific protein or the Petition 870260057806, dated 06 / 15 / 2026, p. 28 / 374 18 / 256 DLL3-binding protein selectively binds to tumor cells that express DLL3. In some modalities, the trispecific DLL3-targeting protein mediates T-cell death of DLL3-expressing tumor cells. In some modalities, the tumor disease comprises a solid tumor disease. In some modalities, the solid tumor disease comprises lung cancer, gastric cancer, ovarian cancer, or triple-negative breast cancer. In some modalities, the solid tumor disease is metastatic.

[0035] An embodiment provides a method for the treatment or improvement of a proliferative disease, or a tumor disease, comprising the administration of a trispecific protein targeting DLL3 comprising a DLL3-binding domain comprising the sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886, or a DLL3-binding protein comprising a sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886. In some embodiments, the trispecific protein targeting DLL3 or the DLL3-binding protein selectively binds to tumor cells expressing DLL3. In some embodiments, the trispecific protein targeting DLL3 directs T-cell death of tumor cells expressing DLL3. In some embodiments, the tumor 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 forms, solid tumor disease is metastatic.

[0036] One embodiment provides a method for the treatment or improvement of a proliferative disease, or a tumorous disease, comprising the administration of a DLL3-binding domain comprising a sequence as presented in SEQ ID No. 68 or 75 or a DLL3-binding protein comprising a sequence as presented in SEQ ID No. 68 or 75. In some embodiments, the trispecific protein targeting DLL3 or the binding protein Petition 870260057806, dated 06 / 15 / 2026, p. 29 / 374 19 / 256 DLL3 at a dose of up to 10 mg / kg. In some modalities, the protein is administered at least once a week. In some modalities, the protein is administered twice a week. In some modalities, the protein is administered bi-weekly. In some modalities, the protein is administered every three weeks.

[0037] In one embodiment, a DLL3-binding protein is provided comprising an amino acid sequence as presented in SEQ ID No. 1890 or SEQ ID No. 1891.

[0038] One embodiment provides a trispecific protein that targets DLL3, wherein said protein comprises (a) a first domain (A) that is a variable single-stranded fragment that specifically binds to a human CD3; (b) a second domain (B) that is a single-domain antibody that specifically binds to a human serum albumin protein; and (c) a third domain (C) that is a single-domain antibody that specifically binds to a DLL3 protein, wherein the third domain (C) comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886.

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

[0040] In some embodiments, the third domain (C) is an affinity-matured binding molecule derived from a parent molecule that specifically binds to the DLL3 protein. In some embodiments, the affinity-matured DLL3-binding molecule has a binding affinity with the Petition 870260057806, dated 06 / 15 / 2026, p. 30 / 374 20 / 256 DLL3 protein which is about 2 times to about 50 times greater than the binding affinity of the parent molecule for the DLL3 protein.

[0041] In some embodiments, the third domain (C) comprises a sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886. In some embodiments, the third domain (C) comprises a sequence that is selected from the group consisting of SEQ ID Nos. 1-52. In some embodiments, the third domain (C) comprises a CDR1, a CDR2, and a CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 443, 884, and 1887, wherein CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 885-1326 and 1888, and wherein CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 495-528. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 937-970.In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1379-1412. In some embodiments, the third domain (C) comprises a sequence selected from the group consisting of SEQ ID Nos. 5386.

[0042] In some embodiments, the third domain (C) comprises a CDR1, a CDR2, and a CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 529-809. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 971 to 1251. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1379-1412. In some embodiments, the third domain (C) comprises a sequence selected from the Petition 870260057806, dated 06 / 15 / 2026, p. 31 / 374 21 / 256 from the group consisting of SEQ ID Nos. 87-367. In some embodiments, the third domain (C) comprises a CDR1, a CDR2, and a CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 810884. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 1252 to 1326. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1692 to 1768. In some embodiments, the third domain (C) comprises a sequence selected from the group consisting of SEQ ID Nos. 368-442. In some embodiments, the first domain (A) comprises a sequence selected from the group consisting of SEQ ID Nos. 1793 to 1807 and 1897-1898.

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

[0044] One embodiment provides a trispecific protein targeting DLL3, wherein said protein comprises (a) a first domain (A) which is a variable single-chain fragment that specifically binds to a human CD3; (b) a second domain (B) which is a single-domain antibody that specifically binds to a human serum albumin protein; and (c) a third Petition 870260057806, dated 06 / 15 / 2026, p. 32 / 374 22 / 256 domain (C) which is a single-domain antibody that specifically binds to DLL3, wherein the third domain comprises a sequence of SEQ ID No. 68 or SEQ ID No. 75, or is derived from SEQ ID No. 68 or SEQ ID No. 75. In some embodiments, the third domain (C) is derived from SEQ ID No. 68 or SEQ ID No. 75. In some embodiments, the third domain (C) comprises the sequence of SEQ ID No. 68 or the sequence of SEQ ID No. 75.

[0045] One embodiment provides a DLL3-binding protein comprising the following formula:f1-r1-f2-r2-f3-r3-f4 where, 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 where f1, f2, f3 and f4 are structural residues. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID Nos. 443884 and 1887, CDR2 comprises a selected sequence from the group consisting of SEQ ID Nos.885-1326 and 1888, and CDR3 comprises a selected sequence from the group consisting of SEQ ID Nos. 1327-1768 and 1889.

[0046] One embodiment provides a trispecific protein targeting DLL3, wherein said protein comprises (a) a first domain (A) which is a variable single-stranded fragment that specifically binds to a human CD3; (b) a second domain (B) which is a single-domain antibody that binds specifically to an albumin protein. Petition 870260057806, dated 06 / 15 / 2026, p. 33 / 374 23 / 256 human serum; and (c) a third domain (C) which is a single-domain antibody that specifically binds to a DLL3 protein, wherein the third domain (C) comprises a CDR1 having the amino acid sequence of SEQ ID No. 874, a CDR2 having the amino acid sequence of SEQ ID No. 1316, and a CDR3 having the amino acid sequence of SEQ ID No. 1758.

[0047] One embodiment provides a trispecific protein targeting DLL3, wherein said protein comprises (a) a first domain (A) which is a variable single-stranded fragment that specifically binds to a human CD3; (b) a second domain (B) which is a single-domain antibody that specifically binds to a human serum albumin protein; and (c) a third domain (C) which is a single-domain antibody that specifically binds to DLL3, wherein the third domain comprises 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 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.In some forms, the third domain (C) comprises a sequence selected from the group consisting of SEQ ID Nos. 408, 425, 432, 430, 431 and 1886.

[0048] In some embodiments, the domains are linked in the order of H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, H2N-(C)-(B)-(A)-COOH, or Petition 870260057806, dated 06 / 15 / 2026, p. 34 / 374 24 / 256 H2N-(C)-(A)-(B)-COOH, or by ligands L1 and L2, in the order of H2N(A)-L1-(B)-L2-(C)-COOH, H2N-(A)-L1-(C)-L2-(B)-COOH, H2N-(B)-L1(A)-L2-(C)-COOH, H2N-(B)-L1-(C)-L2-(A)-COOH, H2N-(C)-L1-(B)-L2(A)-COOH, or H2N-(C)-L1-(A)-L2-(B)-COOH.

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

[0050] In some embodiments, the affinity-matured DLL3-binding molecule has a binding affinity for the DLL3 protein that is about 2 times 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 SEQ ID No. 68 or SEQ ID No. 75.

[0051] In some embodiments, the L1 and L2 ligands are each independently selected from the group consisting of (GS)n (SEQ ID No. 1809), (GGS)n (SEQ ID No. 1810), (GGGS)n (SEQ ID No. 1811), (GGSG)n (SEQ ID No. 1812), (GGSGG)n (SEQ ID No. 1813), or (GGGGS)n (SEQ ID No. 1814), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the L1 and L2 ligands independently comprise the sequence GGGGSGGGS (SEQ ID No. 1808).

[0052] In some embodiments, the second domain (B) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 1769-1778. In some embodiments, the first domain (A) comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 1793-1802 and 1897-1898. In some embodiments, the protein Petition 870260057806, dated 06 / 15 / 2026, p. 35 / 374 25 / 256 trispecific targeting DLL3 comprises the SEQ ID No. 1890 or SEQ ID No. 1891 sequence. In some embodiments, the third domain (C) binds to a human DLL3 protein comprising the SEQ ID No. 1893 sequence.

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

[0054] One embodiment provides a 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 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.

[0055] A modality provides a method for treating or improving a disease, comprising administering an effective amount of a trispecific protein targeting DLL3, according to any of the above modalities. Incorporation by Reference

[0056] All publications, patents and patent applications mentioned in this descriptive report are incorporated herein by reference to the same extent as if each individual publication, patent or patent application had been specifically indicated for incorporation by Petition 870260057806, dated 06 / 15 / 2026, page 36 / 374 26 / 256 reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The innovative features of the invention are presented in particular in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description which defines the illustrative embodiments in which the principles of the invention are used, and to the appended drawings, in which: Fig. 1 illustrates the various domains of a trispecific protein targeting DLL3 exemplary of this revelation.

[0058] Fig. 2 illustrates results of a T cell-dependent cellular cytotoxicity (TDCC) assay in DMS-153 cells, using exemplary trispecific proteins targeting DLL3 containing DLL3-binding domains of this revelation, DH18, DH11, DH67 and DH56.

[0059] Fig. 3 illustrates results of a TDCC assay in DMS-153 cells, using exemplary trispecific proteins that target DLL3 containing exemplary DLL3-binding domains of this development DH2, DH43, DH10 and DH6.

[0060] Fig. 4 illustrates results of a TDCC assay in DMS-153 cells, using exemplary trispecific DLL3-targeting proteins containing exemplary DLL3-binding domains of this development DH82, DH23, DH89 and DH17.

[0061] Fig. 5 illustrates results of a TDCC assay in DMS-153 cells, using exemplary trispecific DLL3-targeting proteins containing exemplary DLL3-binding domains of this development DH83, DH12, DH61 and DH29.

[0062] Fig. 6 illustrates results of a TDCC assay in DMS-153 cells, using exemplary trispecific proteins that target DLL3 containing exemplary DLL3-binding domains of this development DH58, and DH70 and a trispecific control protein.

[0063] Fig. 7 illustrates results of a TDCC trial in Petition 870260057806, dated 06 / 15 / 2026, page 37 / 374 27 / 256 DMS-153 cells, using exemplary affinity-mature DLL3-targeting trispecific proteins containing exemplary DLL3-targeting domains of this revelation 1A011, 2E05, 1H012, 2E02 and 1C03.

[0064] Fig. 8 illustrates results of a TDCC assay in DMS-153 cells, using exemplary affinity-mature DLL3-binding trispecific proteins containing exemplary DLL3-targeting domains of this revelation 2E010, 2E01, 2H02, 2A04 and 2F11.

[0065] Fig. 9 illustrates results of a TDCC assay in DMS-153 cells, using exemplary affinity-mature DLL3-binding trispecific proteins containing exemplary DLL3-targeting domains of this revelation 2E011, 3C04, 4H04, 4H011 and 4D09.

[0066] Fig. 10 illustrates results of a TDCC assay in DMS-153 cells, using exemplary affinity-mature DLL3-binding trispecific proteins containing exemplary DLL3-targeting domains of this revelation 4B07, 4E02, 4C06, 3H011 and 3D07.

[0067] Fig. 11 illustrates results of a TDCC assay in DMS-153 cells, using exemplary affinity-mature DLL3-targeting trispecific proteins containing exemplary DLL3-binding domains of this development 3H06, and 4B011, and parental DLL-binding domains DH43, DH6 and a trispecific control protein.

[0068] Fig. 12 illustrates results of a TDCC assay in DMS-153 cells, using exemplary purified affinity-ripened CHO DLL3-binding trispecific proteins containing exemplary DLL3-targeting domains of this development 2E05-M106Y, 2E05-M106Q, 4D09M34L and 4H11-M34L.

[0069] Fig. 13 illustrates results of a TDCC assay in DMS-153 cells, using trispecific proteins that Petition 870260057806, dated 06 / 15 / 2026, pp. 38 / 374 28 / 256 exemplary purified affinity-ripened CHO target DLL3 proteins containing exemplary DLL3-binding domains of this revelation 1A011 (marked as 1A11 in Fig. 13), 1H012 (marked as 1H12 in Fig. 13), 2E02 and 2E05.

[0070] Fig. 14 illustrates results of a TDCC assay in DMS-153 cells, using exemplary purified affinity-ripened CHO trispecific DLL3-target proteins containing exemplary DLL3-binding domains of this revelation 2H02, 3C04, 4D09 and 4H11.

[0071] Fig. 15 illustrates results of a TDCC assay in DMS-153 cells, using purified exemplary trispecific proteins targeting DLL3 containing exemplary parental DLL3-binding domains DH43 and DH6, and a trispecific control protein targeting GFP.

[0072] Fig.Figure 16 illustrates results from a TDCC DMS-153 cell assay, using exemplary trispecific proteins targeting DLL3 containing exemplary DLL3-binding domains, demonstrating this development from the second affinity maturation cycle.

[0073] Fig. 17 illustrates an SDS-PAGE image of 10-20% TRIS Glycine loaded with 2.4 micrograms of unreduced protein per strip and stained with Coomassie. Strip numbers are indicated by the numbers at the top of the gel image and the migration of molecular weight patterns are indicated by the number on the right side of the gel image (in kilodaltons). Gel load: Lane 1 empty, lane 2 molecular weight standard, lane 3 empty, lane 4 trispecific anti-DLL3 containing DLL3 binding domain 51G2, lane 5 trispecific anti-DLL3 containing DLL3 binding domain 51G10, lane 6 trispecific anti-DLL3 containing DLL3 binding domain 51H5, lane 7 trispecific anti-DLL3 containing DLL3 binding domain 51X5, lane 8 trispecific anti-DLL3 containing DLL3 binding domain 52B1, lane 9 trispecific anti-DLL3 containing domain of Petition 870260057806, dated 06 / 15 / 2026, page 39 / 374 29 / 256 link to DLL3 52C4, track 10 trispecific anti-DLL3 containing DLL3 link domain 52D4, track 11 trispecific anti-DLL3 containing DLL3 link domain 51A2, track 12 containing trispecific anti-DLL3 link domain 51A5, track 13 trispecific anti-DLL3 containing DLL3 link domain 51F3, track 14 empty, and track 15 empty.

[0074] Fig. 18 illustrates results of a TDCC assay in DMS-53 cells, using exemplary trispecific DLL3-targeting proteins expressed by purified affinity-ripened CHO containing exemplary DLL3-binding domains of this development 51G2, 51G10, 51H5, 51X5, 52B1, 52C4, 52D4, 51A2, and parental DLL3 binding domain DH6 and a trispecific control protein.

[0075] Fig. 19 illustrates results of a TDCC assay in DMS-153 cells, using exemplary purified affinity-ripened CHO trispecific DLL3-targeting proteins containing exemplary DLL3-binding domains 51G2, 51G10, 51H5, 51X5, 52B1, 52C4, 52D4, 51A2, and the parental DLL3 binding domain DH6, and a trispecific control binding protein targeting GFP.

[0076] Fig. 20 provides a schematic illustration of a trispecific protein targeting DLL3 containing an exemplary DLL3-binding protein (DLL3 ligand), a CD3-binding domain (scFv epsilon anti-CD3), and an albumin-binding domain (anti-ALB), in an anti-DLL3:anti-ALB:anti-CD3 orientation (TAC orientation).

[0077] Fig. 21 provides a schematic illustration of a trispecific protein targeting DLL3 containing an exemplary DLL3-binding protein (DLL3 ligand), a CD3-binding domain (scFv epsilon anti-CD3), and an albumin-binding domain (anti-ALB), in an anti-CD3:anti-ALB:anti-DLL3 orientation (CAT orientation). Petition 870260057806, dated 06 / 15 / 2026, page 40 / 374 30 / 256

[0078] Fig. 22 illustrates results of a T cell-dependent cellular cytotoxicity (TDCC) assay in NCI-H2171 cells, using exemplary trispecific DLL3 proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration or in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) or bovine serum albumin (BSA).

[0079] Fig. 23 illustrates results of a T cell-dependent cellular cytotoxicity (TDCC) assay in DMS-79 cells, using exemplary trispecific DLL3-targeting proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration or in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence or absence of human serum albumin (HSA).

[0080] Fig. 24 illustrates results of a T cell-dependent cellular cytotoxicity (TDCC) assay in SHP77 cells, using exemplary trispecific DLL3 proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration or in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) or bovine serum albumin (BSA).

[0081] Fig. 25 illustrates results of a T cell-dependent cellular cytotoxicity (TDCC) assay in WM2664 cells, using exemplary trispecific DLL3 proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration or in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA) or bovine serum albumin (BSA).

[0082] Fig. 26 depicts the binding of trispecific proteins Petition 870260057806, dated 06 / 15 / 2026, page 41 / 374 31 / 256 that target exemplary DLL3 containing DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration for human T cells from four different donors compared to that of a control with secondary antibody alone or cells without any antibody or trispecific molecule.

[0083] Fig. 27 depicts the binding of exemplary trispecific proteins targeting DLL3 containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration to human T cells from four different donors compared to that of a control with secondary antibody alone or cells without any antibody or trispecific molecule.

[0084] Fig. 28 depicts the binding of exemplary trispecific proteins targeting DLL3 containing a DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration for human DLL3 expressing cell lines NCI-H82 (top left), SHP77 (top right), DMS53 (bottom left) or NCI-H2171 (bottom right) compared to trispecific molecules with a GFP-binding domain.

[0085] Fig. 29 depicts the binding of exemplary trispecific DLL3-targeting proteins containing a DLL3-binding domain of this revelation, 52D04, in an antiCD3:anti-ALB:anti-DLL3 (CAT) configuration to human DLL3 expressing cell lines NCI-H82 (top left), SHP77 (top right), DMS53 (bottom left) or NCI-H2171 (bottom right) compared to trispecific molecules with a GFP-binding domain.

[0086] Fig. 30 illustrates the results of a TDCC assay in NCI-H82 cells, using exemplary trispecific proteins that target DLL3 containing a DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:anti configuration. Petition 870260057806, dated 06 / 15 / 2026, page 42 / 374 32 / 256 ALB:anti-CD3 (TAC) antibodies were tested in the presence of human serum albumin (HSA) using T cells from four different donors.

[0087] Fig. 31 illustrates the results of a TDCC assay in SHP77 cells, using exemplary trispecific DLL3-targeting proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:antiALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors.

[0088] Fig. 32 illustrates the results of a TDCC assay in DMS53 cells, using exemplary trispecific DLL3-targeting proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:antiALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors.

[0089] Fig. 33 illustrates the results of a TDCC assay in NCI-H2171 cells, using exemplary trispecific DLL3-targeting proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:antiALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors.

[0090] Fig. 34 illustrates the results of a TDCC assay in NCI-H82 cells, using exemplary trispecific DLL3-targeting proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors.

[0091] Fig. 35 illustrates the results of a TDCC assay in SHP77 cells, using trispecific proteins that Petition 870260057806, dated 06 / 15 / 2026, page 43 / 374 33 / 256 exemplary DLL3 targets containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:antiALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors.

[0092] Fig. 36 illustrates the results of a TDCC assay in DMS53 cells, using exemplary trispecific DLL3-targeting proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors.

[0093] Fig. 37 illustrates the results of a TDCC assay in NCI-H2171 cells, using exemplary trispecific DLL3-targeting proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors.

[0094] Fig. 38 illustrates the results of flow cytometry measurements of CD69 expression in T cells cocultured with NCI-H82 cells with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors.

[0095] Fig. 39 illustrates the results of flow cytometry measurements of CD25 expression in T cells co-cultured with NCI-H82 cells with a titration of exemplary DLL3-targeting trispecific proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in Petition 870260057806, dated 06 / 15 / 2026, page 44 / 374 34 / 256 presence of human serum albumin (HSA), using T cells from four different donors.

[0096] Fig. 40 illustrates the results of flow cytometry measurements of CD69 expression in T cells cocultured with DMS53 cells with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors.

[0097] Fig. 41 illustrates the results of flow cytometry measurements of CD25 expression in T cells cocultured with DMS53 cells with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0098] Fig. 42 illustrates the results of flow cytometry measurements of CD69 expression in T cells cocultured with NCI-H82 cells with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors.

[0099] Fig. 43 illustrates the results of flow cytometry measurements of CD25 expression in T cells cocultured with NCI-H82 cells with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors. Petition 870260057806, dated 06 / 15 / 2026, page 45 / 374 35 / 256

[0100] Fig. 44 illustrates the results of flow cytometry measurements of CD69 expression in T cells cocultured with DMS53 cells with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors.

[0101] Fig. 45 illustrates the results of flow cytometry measurements of CD25 expression in T cells cocultured with DMS53 cells with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA).

[0102] Fig. 46 illustrates the results of IFNγ measurements in conditioned media of co-cultures of T cells and NCIH82 cells incubated with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:antiALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0103] Fig. 47 illustrates the results of IFNγ measurements in conditioned media of co-cultures of T cells and SHP77 cells incubated with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:antiALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0104] Fig. 48 illustrates the results of IL-2 measurements in conditioned media of co-cultures of T cells and NCIH82 cells incubated with a titration of exemplary trispecific DLL3-targeting proteins containing a binding domain. Petition 870260057806, dated 06 / 15 / 2026, page 46 / 374 36 / 256 DLL3 of this discovery, 52D04, in an anti-DLL3:antiALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0105] Fig. 49 illustrates the results of IL-2 measurements in conditioned media of co-cultures of T cells and SHP77 cells incubated with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:antiALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0106] Fig. 50 illustrates the results of TNFα measurements in conditioned media of co-cultures of T cells and NCIH82 cells incubated with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:antiALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0107] Fig. 51 illustrates the results of TNFα measurements in conditioned media of co-cultures of T cells and SHP77 cells incubated with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:antiALB:anti-CD3 (TAC) configuration, tested in the presence of human serum albumin (HSA).

[0108] Fig. 52 illustrates the results of IFNγ measurements in conditioned media of co-cultures of T cells and NCIH82 cells incubated with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA).

[0109] Fig. 53 illustrates the results of IFNγ measurements in conditioned media of co-cultures of T cells and SHP77 cells. Petition 870260057806, dated 06 / 15 / 2026, page 47 / 374 37 / 256 incubated with a titration of exemplary trispecific proteins targeting DLL3 containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA).

[0110] Fig. 54 illustrates the results of IL-2 measurements in conditioned media of co-cultures of T cells and NCIH82 cells incubated with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA).

[0111] Fig. 55 illustrates the results of IL-2 measurements in conditioned media of co-cultures of T cells and SHP77 cells incubated with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA).

[0112] Fig. 56 illustrates the results of TNFα measurements in conditioned media of co-cultures of T cells and NCIH82 cells incubated with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:antiALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA).

[0113] Fig. 57 illustrates the results of TNFα measurements in conditioned media of co-cultures of T cells and SHP77 cells incubated with a titration of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA). Petition 870260057806, dated 06 / 15 / 2026, page 48 / 374 38 / 256

[0114] Fig. 58 depicts exemplary trispecific DLL3-targeting proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:antiALB:anti-CD3 (TAC) configuration or an anti-CD3:anti-ALB:antiDLL3 (CAT) configuration, had the ability to inhibit tumor growth in mice injected with a mixture of human T cells and NCI-H82 small cell lung cancer cells at dosages of 20 pg / kg, 100 pg / kg or 500 pg / kg.

[0115] Fig. 59 depicts exemplary trispecific DLL3-targeting proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:antiALB:anti-DLL3 (CAT) configuration, had the ability to eliminate NCI-H82 xenograft tumor growth in mice injected with human T cells at dosages of 10 pg / kg and 100 pg / kg.

[0116] Fig. 60 depicts exemplary trispecific DLL3-targeting proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-CD3:antiALB:anti-DLL3 (CAT) configuration, having the ability to inhibit tumor growth in mice injected with a mixture of human T cells and SHP77 small cell lung cancer cells at dosages of 10 pg / kg and 100 pg / kg.

[0117] Fig. 61 depicts the pharmacokinetic profile of exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration (ID numbers 1 and 2) or an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration (ID numbers 3 and 4). Serum levels of DLL3-targeting trispecific proteins at various time points after injection into cynomolgus monkeys at 0.3 mg / kg are shown in the graph.

[0118] Fig. 62 depicts the pharmacokinetic profile of exemplary trispecific proteins targeting DLL3 containing DLL3-binding domain of this revelation, 52D04, in a Petition 870260057806, dated 06 / 15 / 2026, page 49 / 374 The graph shows the 39 / 256 anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration at various time points after injection in cynomolgus monkeys at 1 mg / kg or 10 mg / kg.

[0119] Fig. 63 depicts the transient cytokine increase after the first dosage of a DLL3-binding TriTAC molecule exemplifying this revelation at 1 mg / kg and 10 mg / kg or a vehicle control. The top panel shows a transient increase in IFNγ, the second panel shows a transient increase in IL-6, and the third panel shows a transient increase in IL-10.

[0120] Fig. 64 illustrates the results of a TDCC assay in DMS53 cells, using exemplary DLL3-targeting trispecific proteins containing DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, using freshly thawed protein, or using protein present in a serum sample from a cynomolgus monkey collected 168 h after assay with 10 mg / kg of DLL3-targeting trispecific protein, measured in the presence of 8.4% cynomolgus monkey serum. DETAILED DESCRIPTION OF THE INVENTION

[0121] Described in this document, in some embodiments, are proteins that specifically bind to delta-like ligand 3 (DLL3) and multispecific (e.g., trispecific) proteins containing the same pharmaceutical compositions, as well as nucleic acids, recombinant expression vectors, and host cells to produce such proteins. Methods are also provided for the use of at least one of the following: the disclosed DLL3-binding proteins, or trispecific DLL3-targeting proteins containing the same, in the prevention and / or treatment of diseases, conditions, and disorders. The trispecific DLL3-targeting proteins have the ability to specifically bind to DLL3 as well as CD3 and have a half-life extension domain, as well as a domain that has the ability to specifically bind to human albumin (ALB). Fig. 1 depicts a Petition 870260057806, dated 06 / 15 / 2026, p. 50 / 374 40 / 256 Non-limiting example of a trispecific DLL3-binding protein. In some embodiments, the trispecific protein targeting DLL3 comprises an antibody, such as a trispecific antibody. Certain definitions

[0122] An antibody typically refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Human light chains comprise a variable (VL) domain and a constant (CL) domain, where the constant domain can be readily classified as kappa or lambda based on amino acid sequence and gene locations. Each heavy chain comprises a variable (VH) domain and a constant region, which, in the case of IgG, IgA, and IgD, comprises three domains designated CH1, CH2, and CH3 (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 (generally from about 10 to about 60 amino acids in IgG).The variable domains in both the light and heavy chains are linked 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 antibody class further comprises intra-chain and inter-chain disulfide bonds formed by paired cysteine ​​residues. There are two types of native disulfide bridges or bonds in immunoglobulin molecules: inter-chain and intra-chain disulfide bonds. The location and number of inter-chain disulfide bonds vary according to the immunoglobulin class and species. Inter-chain disulfide bonds are located on the surface of the immunoglobulin, are solvent-accessible, and are often relatively reduced. Petition 870260057806, dated 06 / 15 / 2026, page 51 / 374 41 / 256 easy. In the human IgG1 isotype there are four disulfide linkages between chains, one for each heavy chain to light chain and two between the heavy chains. The disulfide linkages between chains are not required for chain association. As is well known, the cysteine-rich hinge region of IgG1 heavy chain has generally been retained to consist of three parts: an upper hinge, a middle hinge, and a lower hinge. Those skilled in the art will observe that the hinge region of IgG1 contains the cysteines in the heavy chain comprising the disulfide linkages between chains (two heavy / heavy, two heavy / light), which provide structural flexibility that facilitates Fab movements. The disulfide linkage between the light and heavy chains of IgG1 is formed between C214 of the kappa or lambda light chain and C220 in the upper hinge region of the heavy chain.The disulfide linkages between the heavy chains are at positions C226 and C229 (all numbered using the EU index according to Kabat, et al., below).

[0123] As used in this document, the term antibody includes polyclonal antibodies, multiclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intrabody antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including mutains 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 Fvs (sdFv), an Fd fragment 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 others Petition 870260057806, dated 06 / 15 / 2026, page 52 / 374 42 / 256 modifications, and any other immunoreactive molecule, provided it comprises a domain having a binding site for association or preferential binding with a DLL3 protein. Furthermore, unless otherwise indicated by contextual restrictions, the term additionally includes all antibody classes (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Constant heavy chain domains corresponding to different antibody classes are typically denoted by the corresponding lowercase Greek letters alpha, delta, epsilon, gamma, and mu, respectively. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0124] In some embodiments, the DLL3-binding domain of the trispecific DLL3-targeting proteins of this development comprises a single antibody heavy chain, such as a VH domain or a VHH domain. In some cases, the DLL3-binding proteins comprise a heavy chain-only antibody that is a manipulated human VH domain. In some instances, the manipulated human VH domain is produced by moving phage display libraries. In some embodiments, the DLL3-binding domain of the trispecific DLL3-targeting proteins of this development comprises a VHH. The term VHH, as used herein, refers to a single-chain antibody-binding domain devoid of light chains. In some cases, a VHH is derived from an antibody type that can be found in Camelidae or cartilaginous fish that is naturally devoid of light chains or from a non-immunized and synthetic VHH that can be constructed accordingly.Each heavy chain comprises a variable region encoded by exons V, D, and J. A VHH, in some cases, is a natural VHH. Petition 870260057806, dated 06 / 15 / 2026, page 53 / 374 43 / 256 as a VHH derived from camelids or a recombinant protein comprising a variable heavy chain domain. In some embodiments, the VHH is derived from a species selected from the group consisting of camels, llamas, vicuñas, 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 breed alpaca or a Suri breed alpaca).

[0125] As used in this document, the term “variable region” or “variable domain” refers to the fact that certain portions of variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not equally derived across all antibody variable domains. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both heavy chain (VH) and light chain (VL) variable domains. The most highly conserved portions of variable domains are called the framework (FR). Native light and heavy chain variable domains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops that connect and, in some cases, form part of the β-sheet framework.The CDRs in each chain are held together in close proximity by the FR regions and, along with the CDRs of the other chain, contribute to the formation of the antibody antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as antibody cleavage in antibody-dependent cellular toxicity. scFv fragments. Petition 870260057806, dated 06 / 15 / 2026, page 54 / 374 44 / 256 (for variable single-chain fragment), which in some cases are obtained by genetic modification, associate into a single polypeptide chain, in the VH region and the VL region of an antibody, separated by a peptide linker.

[0126] In some embodiments of this disclosure, the DLL3-binding domain, such as the DLL3-binding domain of trispecific proteins targeting DLL3, comprises a single-domain antibody, such as single heavy-chain antibodies, such as VH or VHH domains, and comprises three CDRs. Such single heavy-chain antibodies, in some embodiments, bind DLL3 as a dimerization-independent monomer with a VL (variable light chain) region for optimal binding affinity. In some embodiments of this disclosure, the CD3-binding domain of trispecific proteins targeting DLL3 comprises an scFv. In some embodiments of this disclosure, the albumin-binding domain of trispecific proteins targeting DLL3 comprises a single heavy-chain antibody, such as a single-domain antibody comprising a VH domain or a VHH domain.

[0127] The assignment of amino acids to each domain, structure region and CDR is, in some embodiments, in accordance with one of the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Edition), US Department 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) unless otherwise noted. The CDRs of this disclosure are not intended to necessarily correspond to the Kabat numbering convention. In some embodiments of this disclosure, the DLL3-binding proteins comprise single-domain antibodies, such as single heavy-chain antibodies, such as VH domains. Petition 870260057806, dated 06 / 15 / 2026, page 55 / 374 45 / 256 or VHH, and comprise three CDRs. Such single heavy chain antibodies, in some embodiments, bind DLL3 as a monomer without dimerization dependence with a VL (variable light chain) region for optimal binding affinity.

[0128] Variable domain residue numbering as in Kabat or amino acid position numbering as in Kabat and variations thereof, refers to the numbering system used for variable heavy chain domains or variable light chain domains of the antibody compilation in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md, USA. (1991). With the use of this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids that correspond to a shortening of, or insertion into, a variable domain FR or CDR. For example, a variable heavy chain domain may include a single amino acid insert (residue 52a according to Kabat) after the H2 residue 52 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after the heavy chain FR residue 82.The Kabat numbering of residues can be determined for a given antibody by aligning homology regions of the antibody sequence with a standard Kabat numbered sequence.

[0129] The term “Structure” or “FR” residues (or regions) refers to variable domain residues other than hypervariable region or CDR residues as defined herein. A “human consensus structure” is a structure that represents the most commonly occurring amino acid residue in a selection of human immunoglobulin VH or VL structure sequences.

[0130] As used in this document, the term “percent (%) of amino acid sequence identity” with respect to a sequence is defined as the percentage of amino acid residues Petition 870260057806, dated 06 / 15 / 2026, page 56 / 374 46 / 256 in a candidate sequence that are identical to the amino acid residues in the specific sequence, after sequence alignment and introduction of gaps if necessary, to achieve the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved in several ways that are included in the skill of the person skilled in the art, for example, with the use of publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Persons skilled in the art can determine appropriate parameters to measure alignment, which includes any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared.

[0131] As used in this document, the term “elimination half-life” is used in its common sense, as described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 2125 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is intended to encompass a quantitative measure of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first-order kinetics), since drug concentrations generally do not approach those required for saturation of the elimination process. The rate of an exponential process can be expressed by its rate constant, k, which expresses the fractional change per unit time, or by its half-time, t1 / 2, the time required for 50% completion of the process. The units of these two constants are time-1 and time, respectively.A first-order constant rate and the reaction half-time are simply related (kxt1 / 2=0.693) and can consequently be interchanged. Since the... Petition 870260057806, dated 06 / 15 / 2026, page 57 / 374 47 / 256 First-order elimination kinetics indicate that a constant fraction of drug is lost per unit time; a plot of the log of drug concentration versus time is linear at all times following the initial distribution phase (i.e., after drug absorption and distribution are complete). The half-life for drug elimination can be precisely determined from such a graph.

[0132] As used in this document, the term “binding affinity” refers to the affinity of the proteins described in the disclosure to their binding targets and is expressed numerically using “Kd” values. If two or more proteins are indicated to have comparable binding affinities to their binding targets, then the Kd values ​​for the binding of the respective proteins to their binding targets are within ±2 times each. If two or more proteins are indicated to have comparable binding affinities to a single binding target, then the Kd values ​​for the binding of the respective proteins to said single binding target are within ±2 times each. If a protein is indicated to bind two or more targets with comparable binding affinities, then the Kd values ​​for the binding of said protein to the two or more targets are within ±2 times each. In general, a higher Kd value corresponds to a weaker binding.In some embodiments, “Kd” is measured by a radiolabeled antigen binding assay (RIA) or surface plasmon resonance assays using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ, USA). In certain embodiments, an “at-rate” or “association rate” or “association rate” or “kon” term and an “off-rate” or “dissociation rate” or “dissociation rate” or “koff” term are also determined with the surface plasmon resonance technique using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ, USA). In additional embodiments, the terms “Kd”, “kon”, and “koff” are measured using the... Petition 870260057806, dated 06 / 15 / 2026, page 58 / 374 48 / 256 OCTET® Systems (Pall Life Sciences). In an exemplary method for measuring binding affinity using OCTET® Systems, the ligand, for example, human DLL3 or biotinylated cynomolgus, is immobilized on the tip surface of an OCTET® streptavidin capillary sensor wherein the streptavidin tips are then activated according to the manufacturer's instructions using approximately 20-50 pg / ml of human DLL3 or cynomolgus protein. A PBS / Casein solution is also introduced as a blocking agent. For binding kinetics measurements, DLL3-binding protein variants are introduced at a concentration in the range of approximately 10 ng / ml to approximately 100 pg / ml, approximately 50 ng / ml to approximately 5 pg / ml, or approximately 2 ng / ml to approximately 20 pg / ml. In some embodiments, single-domain DLL3-binding proteins are used at a concentration in the range of about 2 ng / ml to about 20 pg / ml.Complete dissociation is observed in the case of the negative control, an assay buffer without the binding proteins. The kinetic parameters of the binding reactions are then determined using an appropriate tool, for example, ForteBio software.

[0133] 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 this 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. It is contemplated that in some embodiments the DLL3-binding protein of this disclosure is reasonably small and not greater than 25 kD, not greater than 20 Petition 870260057806, dated 06 / 15 / 2026, p. 59 / 374 49 / 256 kDa, not greater than 15 kDa, or not greater than 10 kDa in some embodiments. In certain examples, the EGFR binding is 5 kDa or less if it is a peptide or a small molecule entity.

[0134] In one aspect, the trispecific DLL3-targeting protein (also referred to herein as a trispecific DLL3-binding protein, a trispecific DLL3 protein, or a TriTAC™ DLL3) 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 trispecific DLL3-targeting proteins are arranged in any order. Thus, the domain order of trispecific DLL3-targeting proteins is contemplated as follows: H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, H2N-(C)-(B)-(A)-COOH or H2N-(C)-(A)-(B)-COOH.

[0135] In some embodiments, trispecific proteins targeting DLL3 have an H2N-(A)-(B)-(C)COOH domain order. In some embodiments, trispecific proteins targeting DLL3 have an H2N-(A)-(C)-(B)-COOH domain order. In some embodiments, trispecific proteins targeting DLL3 have an H2N-(B)-(A)-(C)-COOH domain order. In some embodiments, trispecific proteins targeting DLL3 have an H2N-(B)-(C)-(A)-COOH domain order. In some embodiments, trispecific proteins targeting DLL3 have an H2N-(C)-(B)-(A)-COOH domain order. In some embodiments, trispecific proteins targeting DLL3 have an H2N-(C)-(A)-(B)-COOH domain order.

[0136] In some embodiments, trispecific proteins Petition 870260057806, dated 06 / 15 / 2026, p. 60 / 374 50 / 256 targeting DLL3 have the HSA-binding domain (also referred to herein as ALB) as the intermediate domain, such that the domain order is either H2N-(A)-(B)(C)-COOH or H2N-(C)-(B)-(A)-COOH. It is contemplated that in such embodiments where the HSA-binding domain is the intermediate domain, the CD3 and DLL3-binding domains are produced with additional flexibility to bind to their respective targets.

[0137] In some embodiments, the trispecific binding protein comprises a third domain that specifically binds to DLL3, wherein the third domain is in some cases a DLL3-binding single-domain antibody that binds to DLL3 with equivalent or better affinity than that of a reference DLL3-binding parent molecule. The third domain in some embodiments comprises an affinity-matured DLL3-binding molecule (e.g., an affinity-matured DLL3-binding single-domain antibody), and is derived from the DLL3-binding parent molecule, which comprises one or more amino acid mutations (e.g., a stabilizing mutation, a destabilizing mutation) relative to the DLL3-binding parent molecule. In some embodiments, the affinity-matured DLL3-binding molecule has superior stability relative to selected destabilizing agents, such as that of a reference DLL3-binding parent molecule.In some embodiments, the affinity-matured DLL3-binding molecule is identified in a process comprising positioning one or more pre-candidate DLL3-binding molecules derived from one or more parental DLL3-binding molecules, expressed in a phage display library, against a DLL3 protein, such as a human DLL3 protein. The pre-candidate DLL3-binding molecule comprises, in some embodiments, amino acid substitutions in the variable regions, CDRs, or framework residues, with... Petition 870260057806, dated 06 / 15 / 2026, page 61 / 374 51 / 256 relative to a parent molecule.

[0138] As used in this document, “Phage display” refers to a technique whereby variant polypeptides are displayed as fusion proteins to at least a portion of a coat protein on the surface of phage, filamentous phage particles. One utility of phage display is that large libraries of randomized protein variants can be rapidly and effectively selected for those sequences that bind to a target molecule with high affinity. Phage peptide and protein library display has been used to screen millions of polypeptides for those with specific binding properties. Polyvalent phage display methods have been used to display small random peptides and small proteins via fusions to the IΠ gene 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 a gene III or a portion thereof, and expressed at low levels in the presence of wild-type gene III protein, so that phage particles display either one copy or no copy of the fusion proteins. Avidity effects are reduced with respect to polyvalent phages, so selection is based on intrinsic ligand affinity, and phagemid vectors are used, which simplify DNA manipulations. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).

[0139] In some embodiments, positioning comprises using varying binding times and concentrations to identify DLL3-binding molecules with increased or decreased association rates from precandidate DLL3-binding molecules. In some embodiments, positioning comprises using varying washing times to identify DLL3-binding molecules with increased or decreased dissociation rates. Petition 870260057806, dated 06 / 15 / 2026, page 62 / 374 52 / 256 of pre-candidate DLL3 molecules. In some embodiments, placement involves using both linkage times variants and wash-off times variants. In some embodiments, one or more stabilizing mutations are combined to increase the stability of the affinity-matured DLL3-binding molecule, for example, by shuffling to create a second-stage combinatorial library from such mutants and conducting a second placement cycle followed by linkage selection.

[0140] In some embodiments, the affinity-matured DLL3-binding molecule comprises an equivalent or better affinity to a DLL3 protein (such as human DLL3 protein) than that of a parent DLL3-binding molecule, but has reduced transreactivity, or in some embodiments, increased transreactivity, with selected substances, such as ligands, proteins, antigens or the like, other than the DLL3 epitope to which a parent DLL3-binding molecule is specific, or is designed to be specific to. With respect to the latter, an affinity-matured DLL3-binding molecule, in some embodiments, is more successfully tested in animal models if the affinity-matured DLL3-binding molecule reacts with both human DLL3 and the corresponding animal model target, mouse DLL3 or cynomolgus DLL3.In some embodiments, the parental DLL3-binding molecule binds to human DLL3 with an affinity of about 10 nM or less, and to cynomolgus DLL3 with an affinity of about 15 nM or less. In some embodiments, the affinity-matured DLL3-binding molecule, identified after one positioning cycle, binds to human DLL3 with an affinity of about 5 nM or less, and to cynomolgus DLL3 with an affinity of about 7.5 nM or less. In some embodiments, the affinity-matured DLL3-binding molecule, identified after two positioning cycles, binds to human DLL3 with an affinity of about... Petition 870260057806, dated 06 / 15 / 2026, p. 63 / 374 53 / 256 of 2.5 nM or less, and DLL3 cynomolgose with an affinity of about 3.5 nM or less.

[0141] In some embodiments, domain A, domain B, and domain C of the trispecific binding protein of this revelation are independently antigen-specific binding domain polypeptides that specifically bind to targets, such as targets on diseased cells, or targets on other cells that support the diseased state, such as targets on stromal cells that support tumor growth or targets on immune cells that support disease-mediated immunosuppression.In some examples, antigen-specific binding domains include antibodies, single heavy chain antibodies, including single-chain antibodies, Fabs, Fv, T cell receptor-binding domains, ligand-binding domains, receptor-binding domains, domain antibodies, single-domain antibodies, minibodies, nanobodies, pepticobodies, or various other antibody mimics (such as aphimers, aphitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, Kunitz domain-based proteins, avimers, knottins, finomers, darpins, aphibodies, afilins, monobodies, and armadillo repeat protein-based proteins).

[0142] In some embodiments, the trispecific proteins targeting DLL3 described in this document comprise a DLL-binding polypeptide with a sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886, subsequences thereof, and variants thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3-binding polypeptide (i.e., the third domain (C)) with at least 70%–95% or more homology to a sequence selected from SEQ ID Nos. 1-442 and 1886, subsequences thereof, and variants thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3-binding polypeptide (i.e., the third domain (C)) Petition 870260057806, dated 06 / 15 / 2026, p. 64 / 374 54 / 256 domain (C)) with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology to a sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886, subsequences thereof, and variants thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3-binding polypeptide (i.e., the third domain (C)) with at least 70%-95% or more identity to a sequence selected from SEQ ID Nos. 1-442 and 1886, subsequences thereof, and variants thereof. In some embodiments, the trispecific antigen-binding protein comprises a DLL3-binding polypeptide (i.e., the third domain (C)) with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a sequence selected from the group consisting of SEQ ID Nos. 1-442 and 1886, subsequences thereof, and variants thereof.

[0143] The trispecific DLL3-targeting proteins described in this document are designed to enable specific targeting of DLL3-expressing cells by recruiting cytotoxic T cells. In some embodiments, this enhances efficacy compared to ADCC (antibody-dependent cell-mediated cytotoxicity), which uses full-length antibodies directed against a single antigen and lacks the ability to directly recruit cytotoxic T cells. Conversely, by engaging CD3 molecules specifically expressed on these cells, the trispecific DLL3-targeting proteins can crosslink cytotoxic T cells with DLL3-expressing cells in a highly specific manner, thus directing the T cell's cytotoxic potential toward the target cell.The trispecific proteins that target DLL3 described in this document engage cytotoxic T cells by binding to surface-expressed CD3 proteins that form part of the TCR. This involves the simultaneous binding of several antigen-binding proteins. Petition 870260057806, dated 06 / 15 / 2026, page 65 / 374 55 / 256 trispecific DLL3 to CD3 and DLL3 expressed on the surface of particular cells causes T cell activation and mediates subsequent lysis of the particular cell expressing DLL3. Thus, trispecific DLL3-targeting proteins are contemplated to exhibit strong, specific, and effective target cell killing. In some embodiments, the trispecific DLL3-targeting proteins described herein stimulate target cell killing by cytotoxic T cells to eliminate pathogenic cells (e.g., tumor cells expressing DLL3). In some such embodiments, cells are selectively eliminated, thus reducing the potential for toxic side effects.

[0144] The trispecific DLL3-targeting proteins described herein confer additional therapeutic advantages over traditional monoclonal antibodies and other smaller bispecific molecules.In general, the efficacy of recombinant protein pharmaceuticals largely depends on the intrinsic pharmacokinetics of the protein itself. One benefit here is that the trispecific DLL3-targeting proteins described herein have an extended pharmacokinetic elimination half-life due to having a half-life extension domain that specifically binds to a serum albumin protein (e.g., human serum albumin protein, HSA). In this context, the trispecific DLL3-targeting proteins described herein have an extended serum elimination half-time of approximately two, three, five, seven, ten, twelve, or fourteen days in some embodiments. This contrasts with other binding proteins, such as BiTE or DART molecules, which have relatively shorter elimination half-lives.For example, the bispecific scPE-scFv fusion molecule BiTE CD19 x CD3 requires continuous intravenous (IV) infusion due to its short elimination half-time. The longer intrinsic half-times of the trispecific proteins it targets are also different. Petition 870260057806, dated 06 / 15 / 2026, page 66 / 374 56 / 256 DLL3s solve this problem by allowing for increased therapeutic potential such as low-dose pharmaceutical formulations, decreased periodic administration, and / or innovative pharmaceutical compositions.

[0145] The DLL3-targeting trispecific proteins described in this document also have an ideal size for enhanced tissue penetration and distribution. Larger sizes limit or prevent protein penetration or distribution into target tissues. The DLL3-targeting trispecific proteins described in this document avoid this by having a small size that allows for enhanced tissue penetration and distribution. Consequently, the DLL3-targeting trispecific proteins described in this document, in some embodiments, have a size of about 50 kDa to about 80 kDa, about 50 kDa to about 75 kDa, about 50 kDa to about 70 kDa, or about 50 kDa to about 65 kDa. In some embodiments, the size of the DLL3-targeting trispecific protein is less than about 60 kDa.Thus, the size of trispecific proteins targeting DLL3 is advantageous over IgG antibodies, which are around 150 kDa, and the BiTE and DART diabody molecules, which are around 55 kDa, but do not have an extended half-life and are therefore rapidly cleared through the kidney.

[0146] In additional embodiments, the trispecific DLL3-targeting proteins described in this document have an optimal size for enhanced tissue penetration and distribution. In these embodiments, the trispecific DLL3-targeting proteins are constructed to be as small as possible while retaining specificity for their targets. Consequently, in these embodiments, the trispecific DLL3-targeting proteins described in this document have a size of approximately 20 kDa to approximately 40 kDa or approximately 25 kDa to approximately 35 kDa to approximately 40 kDa, approximately 45 kDa, approximately 50 kDa, approximately 55 kDa, approximately 60 kDa, approximately 65 kDa. In some Petition 870260057806, dated 06 / 15 / 2026, p. 67 / 374 57 / 256 embodiments, the trispecific proteins targeting DLL3 described in this document have a size of approximately 50 kDa, 49 kDa, 48 kDa, 47 kDa, 46 kDa, 45 kDa, 44 kDa, 43 kDa, 42 kDa, 41 kDa, 40 kDa, approximately 39 kDa, approximately 38 kDa, approximately 37 kDa, approximately 36 kDa, approximately 35 kDa, approximately 34 kDa, approximately 33 kDa, approximately 32 kDa, approximately 31 kDa, approximately 30 kDa, approximately 29 kDa, approximately 28 kDa, approximately 27 kDa, approximately 26 kDa, approximately 25 kDa, approximately 24 kDa, approximately 23 kDa, approximately 22 kDa, approximately 21 kDa or approximately 20 kDa. An exemplary approach to small size is through the use of single-domain antibody fragments (sdAbs) for each of the domains. For example, a particular trispecific DLL3 antigen-binding protein has an anti-CD3 sdAb, an anti-ALB sdAb, and an sdAb for DLL3. This reduces the size of the exemplary DLL3 trispecific antigen-binding protein to below 60 kDa.Thus, in some embodiments, the domains of the trispecific proteins that target DLL3 are all single-domain antibody fragments (sdAb). It is contemplated that in some embodiments the DLL3-binding protein is reasonably small and not greater than 25 kDa, not greater than 20 kDa, not greater than 15 kDa, or not greater than 10 kDa in some embodiments. In certain examples, the DLL3-binding protein is 5 kDa or less if it is a peptide or a small molecule entity.

[0147] In other embodiments, the trispecific proteins targeting DLL3 described in this document comprise small molecule entity (SME) ligands for ALB, DLL3, CD3, or all of them. SME ligands are small molecules averaging about 500 to 2,000 Da in size and are attached to trispecific proteins targeting DLL3 by known methods such as sortase binding or conjugation. In these examples, one of the DLL3 trispecific antigen-binding protein domains is a sortase recognition sequence, LPETG (SEQ ID No: Petition 870260057806, dated 06 / 15 / 2026, p. 68 / 374 58 / 256 1896). To attach an SME ligand to the DLL3 trispecific antigen-binding protein with a sortase recognition sequence, the protein is incubated with a sortase and an SME ligand wherein the sortase attaches the SME ligand to the recognition sequence. In still other embodiments, the DLL3-binding domain of DLL3-targeting trispecific proteins described in this document comprises a knottin peptide for DLL3 binding. Knottins are disulfide-stabilized peptides with a cysteine ​​knot structure and have average sizes of about 3.5 kDa. Knottins have been contemplated for binding to certain tumor molecules such as DLL3. In further embodiments, the third DLL3-binding domain of DLL3-targeting trispecific proteins described in this document comprises a natural DLL3 ligand.

[0148] Another feature of the trispecific DLL3-targeting proteins described in this document is that they are of a simple polypeptide design with flexible linkage of their domains. This allows for easy production and fabrication of the trispecific DLL3-targeting proteins since they can be encoded by a single cDNA molecule that is easily incorporated into a vector. Furthermore, due to the fact that the trispecific DLL3-targeting proteins described in this document are a single monomeric polypeptide chain, there are no chain pairing problems or a requirement for dimerization. It is considered that the trispecific DLL3-targeting proteins described in this document have a reduced tendency to aggregate, unlike other molecules reported as bispecific proteins with Fc-gamma immunoglobulin domains.

[0149] In the trispecific proteins targeting DLL3 described in this document, the domains are, in some embodiments, linked by internal linkers L1 and L2, where L1 Petition 870260057806, dated 06 / 15 / 2026, p. 69 / 374 L1 / L2 links the first and second domains of trispecific proteins targeting DLL3, and L1 links the second and third domains of trispecific proteins targeting DLL3. The L1 and L2 linkers have optimized length and / or amino acid composition. In some embodiments, the L1 and L2 linkers are of the same length and amino acid composition. In other embodiments, L1 and L2 are different. In certain embodiments, the internal linkers L1 and / or L2 are short, i.e., they consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the internal linkers consist of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide linkage. In certain embodiments, the internal L1 and / or L2 linkers are long, that is, they consist of 15, 20, or 25 amino acid residues.In some embodiments, these internal linkers consist of about 3 to about 15, for example, 8, 9, or 10 contiguous amino acid residues. Regarding the amino acid composition of the L1 and L2 internal linkers, the peptides are selected for properties that confer flexibility to the trispecific proteins targeting DLL3, do not interfere with the binding domains, and resist protease cleavage. For example, glycine and serine residues generally provide protease resistance. Examples of suitable internal linkers for binding the domains in these trispecific proteins targeting DLL3 include, but are not limited to, (GS)n (SEQ ID No. 1809), (GGS)n (SEQ ID No. 1810), (GGGS)n (SEQ ID No. 1811), (GGSG)n (SEQ ID No. 1812), (GGSGG)n (SEQ ID No. 1813), (GGGGS)n (SEQ ID No. 1814), (GGGGG)n (SEQ ID No. 1815), or (GGG)n (SEQ ID No. 1816), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the 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 binder L1 and / or L2 is GGGGSGGGS (SEQ ID No. 1808).

[0150] In some cases, domains in the trispecific protein Petition 870260057806, dated 06 / 15 / 2026, p. 70 / 374 60 / 256 targeting DLL3 are conjugated using a site-specific enzymatic conjugation method involving the use of a mammalian or bacterial transglutaminase enzyme. Microbial transglutaminases (mTGs) are versatile tools in modern research and biotechnology. The availability of large quantities of relatively pure enzymes, ease of use, and absence of regulation by calcium and guanosine-5'-triphosphate (GTP) has driven mTG to be the primary crosslinking enzyme used in both the food industry and biotechnology. Currently, mTGs are used in many applications to attach proteins and peptides to small molecules, polymers, surfaces, DNA, as well as other proteins. (See, Pavel Strp, Veracity of microbial transglutaminase, Bioconjugate Chem. 25, 5, 855-862).

[0151] In some examples, trispecific proteins targeting DLL3 are provided in which one of the domains comprises a glutamine receptor in a constant region, which can then be conjugated to another domain via a lysine-based ligand (e.g., any primary amino chain that is a substrate for TGase, comprising an alkylamine, oxamine) wherein conjugation occurs exclusively on one or more glutamine receptor residues present in the target fraction outside the antigen-binding site (e.g., outside a variable region, in a constant region). Conjugation, therefore, does not occur on a glutamine, a glutamine at least partially exposed to the surface, in the variable region. The trispecific protein, in some examples, is formed by reacting one of the domains with a lysine-based ligand in the presence of a TGase.

[0152] In some embodiments, where one or more domains in the trispecific binding protein targeting DLL3 are directly joined, a hybrid vector is produced in which the DNA encoding the directly joined domains are themselves directly linked to each other. In some embodiments, where the Petition 870260057806, dated 06 / 15 / 2026, p. 71 / 374 61 / 256 linkers are used, a hybrid vector is produced in which the DNA encoding a first domain among the three domains is ligated to the DNA encoding one end of a first linker fragment and the DNA encoding a second domain among the three domains is ligated to the other end of the first linker fragment; furthermore, the DNA encoding the second domain among the three domains is ligated to one end of a second linker fragment and the DNA encoding a third domain among the three domains is ligated to the other end of the second linker fragment, wherein the first domain, the second domain, and the third domain are distinct and wherein the first domain, the second domain, and the third domain are independently selected from domain A, domain B, and domain C. Such ligation is performed, for example, in series or as a three-way ligation. CD3 binding domain

[0153] The specificity of the T cell response is mediated by antigen recognition (displayed in the context of a major histocompatibility complex, MHC) by the TCR. As part of the TCR, CD3 is a protein complex that includes a CD3γ (gamma) chain, a CD3δ (delta) chain, and two CD3ε (epsilon) chains that are present on the cell surface. CD3 associates with the α (alpha) and β (beta) chains of the TCR as well as CD3ζ (zeta) to fully comprise the complete TCR. The clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodies, results in T cell activation similar to T cell receptor binding, but independently of its typical clone specificity.

[0154] In one aspect, the trispecific DLL3-targeting proteins described herein comprise a domain that specifically binds to CD3. In one aspect, the trispecific DLL3-targeting proteins described herein comprise a domain that specifically binds to human CD3. Petition 870260057806, dated 06 / 15 / 2026, p. 72 / 374 62 / 256 In some embodiments, the trispecific DLL3-targeting proteins described in this document comprise a domain that specifically binds to CD3γ. In some embodiments, the trispecific DLL3-targeting proteins described in this document comprise a domain that specifically binds to CD3δ. In some embodiments, the trispecific DLL3-targeting proteins described in this document comprise a domain that specifically binds to CD3s.

[0155] In additional embodiments, the trispecific DLL3-targeting proteins described in this document comprise a domain that specifically binds to the TCR. In certain examples, the trispecific DLL3-targeting proteins described in this document comprise a domain that specifically binds to the α chain of the TCR. In certain examples, the trispecific DLL3-targeting proteins described in this document comprise a domain that specifically binds to the β chain of the TCR.

[0156] In certain embodiments, the CD3-binding domain of the trispecific DLL3-targeting proteins described in this document exhibits not only potent CD3-binding affinities with human CD3, but also shows excellent transreactivity with the respective cynomolgus monkey CD3 proteins.

[0157] In some embodiments, the CD3-binding domain of the DLL3 trispecific antigen-binding protein may be any CD3-binding domain including, but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some instances, it is beneficial for the CD3-binding domain to be derived from the same species in which the DLL3 trispecific antigen-binding protein will ultimately be used. For example, for use in humans, it may be beneficial for the CD3-binding domain of the DLL3 trispecific antigen-binding protein to be derived from the same species in which the CD3-binding protein will ultimately be used. Petition 870260057806, dated 06 / 15 / 2026, page 73 / 374 63 / 256 DLL3 trispecific antigen comprises human or humanized residues of the antigen-binding domain of an antibody or antibody fragment.

[0158] 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 complementary light chain that determines region 1 (LC CDR1), complementary light chain that determines region 2 (LC CDR2), and complementary light chain that determines region 3 (LC CDR3) of a humanized or human anti-CD3 binding domain described herein, and / or one or more (e.g., all three) of the complementary heavy chain that determines region 1 (HC CDR1), complementary heavy chain that determines region 2 (HC CDR2), and complementary heavy chain that determines region 3 (HC CDR3) of a humanized or human anti-CD3 binding domain described herein, a humanized or human anti-CD3 binding domain comprising one or more, all three, LC CDRs and one or more, all three, HC CDRs.

[0159] In some embodiments, the humanized or human antiCD3 binding domain comprises a humanized or human CD3-specific light chain variable region wherein the CD3-specific light chain variable region comprises human or non-human light chain CDRs in a human light chain structure region. In certain examples, the light chain structure region is a λ (lambda) light chain structure. In other examples, the light chain structure region is a κ (kappa) light chain structure.

[0160] In some embodiments, the humanized or human antiCD3 binding domain comprises a humanized or human CD3-specific heavy chain variable region in which the region Petition 870260057806, dated 06 / 15 / 2026, page 74 / 374 64 / 256 CD3-specific heavy chain variable comprises human or non-human heavy chain CDRs in a human heavy chain structure region.

[0161] In certain examples, the regions that determine heavy chain and / or light chain complementarity are derived from known anti-CD3 antibodies, such as, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLBT3.4.2, 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.

[0162] In one embodiment, the anti-CD3 binding domain is a single-chain variable fragment (scFv) comprising a light chain and a heavy chain of an amino acid sequence provided herein. As used herein, “single-chain variable fragment” or scFv refers to an antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the variable light and heavy chain regions are continuously linked via a short, flexible polypeptide linker, and has the ability to be expressed as a single polypeptide chain, and wherein the scFv retains the intact antibody specificity of that from which it is derived.In one embodiment, the anti-CD3 binding domain comprises: a variable light chain region comprising an amino acid sequence with 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 from a variable light chain region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein. Petition 870260057806, dated 06 / 15 / 2026, page 75 / 374 65 / 256 document; and / or a variable heavy chain region comprising an amino acid sequence with 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 from a variable heavy chain region provided in this document, or a sequence with 9599% identity to an amino acid sequence provided in this document. In some examples, the anti-CD3 binding domain comprises a sequence selected from SEQ ID Nos. 1793-1807, or a sequence that has 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 Nos. 1793-1807.In some examples, the anti-CD3 binding domain comprises three heavy-chain CDRs (HC CDR1, HC CDR2, and HC CDR3), and three light-chain CDRs. The heavy-chain CDR1 (HC CDR1) of the CD3 binding domain comprises a sequence selected from SEQ ID Nos. 1820-1831, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID Nos. 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 Nos. 1832-1841, or a sequence comprising one or more modifications or substitutions in a sequence selected from SEQ ID Nos. 1832-1841. The heavy chain CDR3 (HC CDR3) of the CD3-binding domain comprises a selected sequence of SEQ ID Nos.1842-1853, or a sequence comprising one or more modifications or substitutions in a selected sequence of SEQ ID Nos. 1842-1853. The CD3-binding domain CDR1 light chain (CDR1 LC) comprises a sequence. Petition 870260057806, dated 06 / 15 / 2026, p. 76 / 374 66 / 256 selected from SEQ ID Nos. 1852-1864, or a sequence comprising one or more modifications or substitutions in a selected sequence from SEQ ID Nos. 1852-1864. The CD3-binding domain CDR2 light chain (LC CDR2) comprises a selected sequence from SEQ ID Nos. 1865-1877, or a sequence comprising one or more modifications or substitutions in a selected sequence from SEQ ID Nos. 1865-1877. The CD3-binding domain CDR3 light chain (LC CDR3) comprises a selected sequence from SEQ ID Nos. 1878-1884, or a sequence comprising one or more modifications or substitutions in a selected sequence from SEQ ID Nos. 1878-1884.In one embodiment, the humanized or human anti-CD3 binding domain is an scFv, and a variable light chain region comprising an amino acid sequence described herein is attached to a variable heavy chain region comprising an amino acid sequence described herein by means of an scFv linker. The variable light chain region and the variable heavy chain region of an scFv may be in either of the following orientations: variable light chain region-variable heavy chain region of scFv linker or variable heavy chain region-variable light chain region of scFv linker.

[0163] In some examples, scFvs that bind to CD3 are prepared according to known methods. For example, scFv molecules can be produced by linking VH and VL regions using flexible polypeptide ligands. The scFv molecules comprise an scFv ligand (e.g., a Ser-Gly ligand) with optimized length and / or amino acid composition. Consequently, in some embodiments, the length of the scFv ligand is such that the VH or VL domain can intermolecularly associate with the other variable domain to form the CD3 binding site. In certain Petition 870260057806, dated 06 / 15 / 2026, p. 77 / 374 In 67 / 256 embodiments, such scFv ligands are short, that is, they consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the scFv ligands consist of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the scFv ligand is a peptide linkage. In some embodiments, these scFv ligands consist of about 3 to about 15, for example 8, 9, or 10 contiguous amino acid residues. Regarding the amino acid composition of the scFv ligands, peptides are selected to confer flexibility, not interfere with the variable domains, and allow interchain folding to bring the two variable domains together to form a functional CD3 binding site. For example, scFv ligands comprising glycine and serine residues generally provide protease resistance. In some embodiments, the ligands in an scFv comprise both glycine and serine residues.The amino acid sequence of scFv ligands can be optimized, for example, by phage display methods to enhance CD3 binding and scFv production yield. Examples of peptide scFv ligands suitable for linking a variable light domain and a variable heavy domain in an scFv include, but are not limited to, (GS)n (SEQ ID No. 1809), (GGS)n (SEQ ID No. 1810), (GGGS)n (SEQ ID No. 1811), (GGSG)n (SEQ ID No. 1812), (GGSGG)n (SEQ ID No. 1813), (GGGGS)n (SEQ ID No. 1814), (GGGGG)n (SEQ ID No. 1815), or (GGG)n (SEQ ID No. 1816), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the scFv ligand may be (GGGGS)4 (SEQ ID No. 1817) or (GGGGS)3. (SEQ ID No. 1818). In some embodiments, a linker comprises a sequence composed of any combinations of the linkers as presented in SEQ ID Nos. 1809 to 1818, and the length of such a linker is in some examples up to 15 amino acids, or longer than 15 amino acids.Variations in ligand length can retain or enhance activity, resulting in superior efficacy in studies. Petition 870260057806, dated 06 / 15 / 2026, page 78 / 374 68 / 256 of activity.

[0164] In some embodiments, the CD3-binding domain of trispecific antigen-binding protein targeting DLL3 has an affinity for CD3 in cells expressing CD3 with a Kd of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In some embodiments, the CD3-binding domain of trispecific antigen-binding protein targeting DLL3 has an affinity for CD3s, γ, or δ with a Kd of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In additional embodiments, the CD3-binding domain of trispecific antigen-binding protein targeting DLL3 has a low affinity for CD3, i.e., about 100 nM or greater.

[0165] The affinity for CD3 binding can be determined, for example, by the ability of the trispecific antigen-binding protein itself targeting DLL3 or its CD3-binding domain to bind to CD3 coated on an assay plate; displayed on a microbial cell surface; in solution; etc. The binding activity of the trispecific antigen-binding protein itself targeting DLL3 or its CD3-binding domain of the present disclosure to CD3 can be assayed by immobilizing the ligand (e.g., CD3) or the trispecific antigen-binding protein itself targeting DLL3 or its CD3-binding domain to a microsphere, substrate, cell, etc. The agents can be added in a suitable buffer and the binding partners incubated for a period of time at a specified temperature.After washing to remove unbound material, the bound protein can be released using, for example, SDS, buffers with a high pH, ​​and the like, and analyzed, for example, by Surface Plasmon Resonance (SPR). Petition 870260057806, dated 06 / 15 / 2026, page 79 / 374 69 / 256 Half-life extension domain

[0166] Domains that extend the half-life of an antigen-binding domain are contemplated in this document. Such domains are contemplated by including, but not limited to, albumin-binding domains, Fc domains, small molecules, and other half-life extension domains known in the art.

[0167] Human albumin (ALB) (molecular mass of 67 kDa) is the most abundant protein in plasma, present at approximately 50 mg / ml (600 μM), and has a half-life of about 20 days in humans. ALB serves to maintain plasma pH, contribute to colloidal blood pressure, functions as a carrier of various metabolites and fatty acids, and serves as a major drug-transporting protein in plasma.

[0168] Non-covalent association with albumin extends the half-life of short-lived proteins. For example, a recombinant fusion of an albumin-binding domain to a Fab fragment resulted in a 25- and 58-fold in vivo release and a 26- and 37-fold half-life extension when administered intravenously to mice and rabbits, respectively, compared to administration of the Fab fragment alone. In another example, when insulin is acylated with fatty acids to promote albumin binding, a prolonged effect was observed when injected subcutaneously in rabbits or pigs. Together, these studies demonstrate a link between albumin binding and prolonged action.

[0169] In one aspect, the trispecific proteins targeting DLL3 described herein comprise a half-life extension domain, for example, a domain that specifically binds to ALB. In some embodiments, the ALB-binding domain of the trispecific antigen-binding protein targeting DLL3 may be any ALB-binding domain including, but not limited to, antibody domains. Petition 870260057806, dated 06 / 15 / 2026, p. 80 / 374 70 / 256 monoclonal, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some embodiments, the ALB-binding domain is single-chain variable fragments (scFv), single-domain antibody such as a variable heavy chain domain (VH), a variable light chain domain (VL), and a variable domain (VHH) of single-domain antibody derived from camelid, peptide, ligand, or HSA-specific small molecule entity. In certain embodiments, the ALB-binding domain is a single-domain antibody. In other embodiments, the HSA-binding domain is a peptide. In further embodiments, the HSA-binding domain is a small molecule. It is contemplated that the HSA-binding domain of DLL3 trispecific antigen-binding protein is reasonably small and not greater than 25 kDa, not greater than 20 kDa, not greater than 15 kDa, or not greater than 10 kDa in some embodiments.In certain examples, the binding to ALB is 5 kDa or less if it is a peptide or small molecular entity.

[0170] The half-life extension domain of trispecific antigen-binding protein targeting DLL3 provides altered pharmacodynamics and pharmacokinetics of the trispecific antigen-binding protein itself that targets DLL3. As above, the half-life extension domain extends the elimination half-time. The half-life extension domain also alters pharmacodynamic properties, including altered tissue distribution, penetration, and diffusion of the trispecific antigen-binding protein. In some embodiments, the half-life extension domain provides enhanced tissue targeting (including tumor tissue), tissue distribution, tissue penetration, tissue diffusion, and improved efficacy compared to a protein without a half-life extension domain. In one embodiment, therapeutic methods effectively and efficiently utilize a reduced amount of the binding protein. Petition 870260057806, dated 06 / 15 / 2026, page 81 / 374 71 / 256 trispecific antigen, resulting in reduced side effects, such as reduced non-tumor cell cytotoxicity.

[0171] Furthermore, the binding affinity of the half-life extension domain can be selected to target a specific elimination halftime in a particular trispecific antigen-binding protein. Thus, in some embodiments, the half-life extension domain has a high binding affinity. In other embodiments, the half-life extension domain has an intermediate binding affinity. In still other embodiments, the half-life extension domain has a low or marginal binding affinity. Exemplary binding affinities include KD concentrations at 10 nM or less (high), between 10 nM and 100 nM (intermediate), and greater than 100 nM (low). As above, ALB binding affinities are determined by methods known as Surface Plasmon Resonance (SPR).In some embodiments, the ALB-binding domains described herein comprise a single-domain antibody.

[0172] In some embodiments, the half-life extension domain comprises a sequence selected from SEQ ID Nos. 1769-1778, or the 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 Nos. 1769-1778. In some examples, the half-life extension comprises three heavy-chain CDRs (HC CDR1, HC CDR2, and HC CDR3) and three light-chain CDRs. In some examples, the half-life extension comprises three heavy-chain CDRs (HC CDR1, HC CDR2, and HC CDR3) or three light-chain CDRs. The heavy-chain CDR1 (HC CDR1) of the half-life extension domain, in some embodiments, comprises a sequence selected from SEQ ID Nos. 1782-1784, or a sequence comprising one or more modifications or substitutions in a Petition 870260057806, dated 06 / 15 / 2026, p. 82 / 374 72 / 256 selected sequence of SEQ ID Nos. 1782-1784, or at least about 80% to about 99%. The CDR2 heavy chain (HC CDR2) of the half-life extension domain, in some embodiments, comprises a selected sequence of SEQ ID Nos. 1785-1790, or a sequence comprising one or more modifications or substitutions in a selected sequence of SEQ ID Nos. 1785-1790. The CDR3 heavy chain (HC CDR3) of the CD3-binding domain comprises a selected sequence of SEQ ID Nos. 1791 or 1792, or a sequence comprising one or more modifications or substitutions in a selected sequence of SEQ ID Nos. 1791 or 1792. DLL3 linking domain

[0173] DLL3 (also known as Delta-like Ligand 3 or SCDO1) is a member of the Delta-like family of DSL Notch ligands. Representative DLL3 protein orthologs include, but are not limited to, human (Accession No. NP_058637 and NP_982353), chimpanzee (Accession No. XP_003316395), mouse (Accession No. NP_031892), and rat (Accession No. NP_446118). In humans, the DLL3 gene consists of 8 exons spanning 9.5 kbp located on chromosome 19q13. Alternative joining in the last exon results in two processed transcripts, one of 2389 bases (Accession No. NM_016941) and one of 2052 bases (Accession No. NM_203486). The previous transcript encodes a protein of 618 amino acids (Accessory number NP_058637), while the last one encodes a protein of 587 amino acids (Accessory number NP_982353).These two DLL3 protein isoforms share, overall, 100% identity along their extracellular and transmembrane domains, differing only in that the longer isoform contains an extended cytoplasmic end that contains 32 additional residues in the protein's carboxy terminus. The extracellular region of the DLL3 protein comprises six EGF-like domains. Petition 870260057806, dated 06 / 15 / 2026, page 83 / 374 73 / 256 simple DSL domain and the N-terminal domain. In general, EGF domains are recognized 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 at approximately amino acid residues 176-215 and the N-terminal domain at approximately amino acid residues 27-175 of hDLL3. Each of the EGF-like domains, the DSL domain, and the N-terminal domain comprises part of the DLL3 protein, as defined by a distinct amino acid sequence. EGF-like domains are designated, in some embodiments, as EGF1 to EGF6, where EGF1 is closest to the N-terminal portion of the protein.In general, DSL ligands are composed of a series of structural domains: a single N-terminal domain, followed by a conserved DSL domain, multiple epidermal growth factor-like repeats, a transmembrane domain, and a cytoplasmic domain that is not highly conserved across ligands, but one that contains multiple lysine residues that are potential sites for ubiquitination by single E3 ubiquitin ligases. The DSL domain is a degenerate EGF domain that is necessary but not sufficient for interactions with Notch receptors. Additionally, the first two EGF-like repeats of almost all DSL ligands contain a protein sequence motif better known as the DOS domain, which interacts cooperatively with the DSL domain when activating Notch signaling.

[0174] In some embodiments, the trispecific DLL3-binding proteins disclosed from this disclosure are generated, manufactured, genetically modified, or selected to interact with a selected domain, motif, or epitope on a DLL3 protein. In some embodiments, the trispecific DLL3-targeting protein binds to the DLL3 domain and, in some embodiments, binds to an epitope comprising G203, Petition 870260057806, dated 06 / 15 / 2026, p. 84 / 374 74 / 256 R205, P206 in the DSL domain.

[0175] The DLL3-binding domains of the trispecific DLL3-targeting proteins of the present disclosure are, in some embodiments, genetically modified, engineered and / or selected to react with either DLL3 isoform(s) or a single protein isoform or, alternatively, comprise a pan-DLL-binding domain that reacts with or associates with at least one additional DLL family member besides DLL3. In some embodiments, the DLL3-binding domains, such as the DLL3-binding domain, are genetically modified, engineered, and / or selected so that they react with domains (or epitopes therein) that are exhibited by DLL3 only or with domains that are at least somewhat conserved across multiple or all DLL family members.

[0176] In some embodiments, the DLL3-binding domain associates with or binds to a specific epitope, portion, motif, or domain of DLL3. Both DLL3 isoforms incorporate an identical extracellular region comprising at least one N-terminal domain, one DSL (Delta / Serrate / lag-2) domain, and six EGF-like domains (i.e., EGF1-EGF6). Consequently, in certain embodiments, the DLL3-binding domain binds with or associates with the N-terminal domain of DLL3 (amino acids 27-175 in the mature protein), while in other embodiments, the DLL3-binding domain associates with the DSL domain (amino acids 176-215) or epitope therein. In other aspects of the present disclosure, the DLL3-binding domain associates with or binds to a specific epitope located in a particular EGF-like domain of DLL3.In some embodiments, the DLL3-binding domain associates with or binds to an epitope located on EGF1 (amino acids 216-249), EGF2 (amino acids 274-310), EGF3 (amino acids 312-351), EGF4 (amino acids 353-389), EGF5 (amino acids 391-427), or EGF6 (amino acids 429-465). In some embodiments, each of the aforementioned domains. Petition 870260057806, dated 06 / 15 / 2026, p. 85 / 374 75 / 256 mentioned comprise more than one epitope and / or more than one container. In some embodiments, the DLL3 binding domain binds to, reacts to, or associates with the DSL domain or an epitope therein. In other embodiments, the DLL3 binding domain binds to, reacts to, or associates with a particular EGF-like domain or an epitope therein. In some embodiments, the DLL3 binding domain binds to, reacts to, or associates with the N-terminal domain or an epitope therein.

[0177] In some embodiments, the DLL3-binding proteins of this development, such as the DLL3-binding domain of the trispecific proteins of this development, bind to the full-length DLL3 protein or to a fragment thereof, such as epitope-containing fragments in the full-length DLL3 protein, as described above. In some cases, the epitope-containing fragment comprises antigenic or immunogenic fragments and derivatives thereof of the DLL3 protein. The epitope-containing fragments, including antigenic or immunogenic fragments, are, in some embodiments, 12 amino acids or more, 20 amino acids or more, 50 or 100 amino acids or more. The DLL3 fragments, in some embodiments, comprise 95% or more of the total protein length, 90% or more, 75% or 50% or 25% or 10% or more of the total protein length.In some embodiments, DLL3 epitope-containing fragments that include antigenic or immunogenic fragments have the ability to elicit a relevant immune response in a patient. DLL3 derivatives include, in some embodiments, sequence variants in which one or more deletions, insertions, or substitutions (e.g., 1-20 as 15 amino acids, or up to 20% as up to 10% or 5% or 1% by number of amino acids based on total protein length) have been produced for the DLL3 sequence provided in SEQ ID No. 1885 (UniProtKB Accession Q9NYJ7). In some embodiments, the substitutions comprise preservative substitutions. Petition 870260057806, dated 06 / 15 / 2026, p. 86 / 374 76 / 256 DLL3 derivatives and variants, in some instances, have essentially the same biological function as the DLL3 protein from which they are derived. For example, DLL3 derivatives and variants are, in some cases, comparatively antigenic or immunogenic for the protein from which they are derived, have the ligand-binding activity, or the ability to form complexes with active receptors, or preferably both, of the protein from which they are derived, and have the same tissue distribution as DLL3.

[0178] The design of the trispecific DLL3-targeting proteins described herein allows the DLL3-binding domain to be flexible, wherein the DLL3-binding domain can be any type of binding domain, including, but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody.In some embodiments, the DLL3-binding domain is a single-chain variable fragment (scFv), a single-domain antibody such as a variable heavy chain (VH) domain, a variable light chain (VL) domain, and a camelid-derived single-domain variable domain (VHH) antibody. In other embodiments, the DLL3-binding domain is a non-Ig binding domain, i.e., an antibody mimic, such as antikalins, afilins, afibody molecules, aphimers, aphitins, alphabodies, avimers, DARPins, finomers, kunitz domain peptides, and monobodies. In further embodiments, the DLL3-binding domain is a ligand or peptide that binds to or associates with DLL3. In still further embodiments, the DLL3-binding domain is a knottin. In still further embodiments, the DLL3-binding domain is a small molecular entity.

[0179] In some embodiments, the DLL3-binding domain binds to a protein comprising the sequence SEQ ID No. 1885 (UniProtKB Accession Q9NYJ7). In some embodiments, the domain of Petition 870260057806, dated 06 / 15 / 2026, p. 87 / 374 77 / 256 DLL3 binding domain binds to a protein comprising a truncated sequence compared to SEQ ID No. 1885 (UniProtKB Accession Q9NYJ7). In some embodiments, the DLL3 binding domain binds to a protein comprising the sequence SEQ ID No. 1892 or SEQ ID No. 1893 (which is a 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 at amino acids 47-4492 of SEQ ID No. 1892.

[0180] In some embodiments, the DLL3-binding domain is an anti-DLL3 antibody or an antibody variant. As used herein, the term antibody variant refers to variants and derivatives of an antibody described herein. In certain embodiments, amino acid sequence variants of the anti-DLL3 antibodies described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-DLL3 antibodies described herein are contemplated to enhance 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 to the nucleotide sequence encoding the antibody or by peptide synthesis.Such modifications include, for example, deletions of and / or insertions of and / or substitutions of residues within the amino acid sequences of the antibody.

[0181] Any combination of deletion, insertion, and substitution can be made to achieve the final construct, since the final construct possesses the desired characteristics, antigen binding. In certain embodiments, antibody variants that have one or more amino acid substitutions are provided. The sites of interest for mutagenesis of Petition 870260057806, dated 06 / 15 / 2026, page 88 / 374 78 / 256 substitutions include the structure regions and CDRs. Examples of such substitutions are described below. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, retained / enhanced antigen binding, decreased immunogenicity, or enhanced T-cell mediated cytotoxicity (TDCC). Both conservative and non-conservative amino acid substitutions are contemplated for preparing antibody variants.

[0182] In another example of a substitution to create a variant anti-DLL3 antibody, one or more residues of the hypervariable region of a parental antibody are substituted. In general, variants are then selected based on improvements in desired properties compared to a parental antibody, for example, increased affinity, reduced affinity, reduced immunogenicity, increased pH dependence of binding.

[0183] In some embodiments, the DLL3-binding domain of the trispecific protein targeting DLL3 is a single-domain antibody such as a variable heavy chain (VH) domain, a variable domain (VHH) of a llama-derived sdAb, a peptide, a ligand, or a DLL3-specific small molecule entity. In some embodiments, the DLL3-binding domain of the trispecific protein targeting DLL3 described herein is any DLL3-binding domain including, but not limited to, domains of a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In certain embodiments, the DLL3-binding domain is a single-domain antibody. In other embodiments, the DLL3-binding domain is a peptide. In further embodiments, the DLL3-binding domain is a small molecule.

[0184] In general, it should be noted that the term antibody of Petition 870260057806, dated 06 / 15 / 2026, p. 89 / 374 79 / 256 single domain as used in this document in its broadest sense is not limited to a specific biological source or a specific preparation method. Single domain antibodies are antibodies whose complementarity-determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, naturally light chain-deficient antibodies, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain structures other than those derived from antibodies. Single domain antibodies can be any of the art or any future single domain antibodies. Single domain antibodies can be derived from any species including, but not limited to, mouse, human, camel, llama, goat, rabbit, bovine. For example, in some embodiments,Single-domain antibodies of revelation are obtained: (1) by isolating the VHH domain from a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by humanizing a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (4) by camelizing a naturally occurring VH domain from any animal species, and in particular from a mammalian species, such as a human, or by expressing a nucleic acid encoding such a camelized VH domain; (5) by camelizing an antibody or Dab domain, or by expressing a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques to prepare proteins,polypeptides or other amino acid sequences; (7) preparing a nucleic acid encoding a single-domain antibody using techniques for nucleic acid synthesis known in the field, followed by expression of the nucleic acid thus obtained; and / or (8) by any, Petition 870260057806, dated 06 / 15 / 2026, page 90 / 374 80 / 256 combination of one or more of the previously mentioned options.

[0185] In one embodiment, a single-domain antibody corresponds to the VHH domains of naturally occurring heavy-chain antibodies directed against DLL3. As further described herein, such VHH sequences can generally be generated or obtained by suitably immunizing a Llama species with DLL3 (i.e., so as to exacerbate an immune response and / or heavy-chain antibodies directed against DLL3), by obtaining a suitable biological sample from said Llama (such as a blood sample, serum sample, or B-cell sample), and by generating DLL3-directed VHH sequences, starting from the example type, using any suitable technique known in the field.

[0186] In another embodiment, such naturally occurring VHH domains against DLL3 are obtained from virgin libraries of Camelid VHH sequences, for example, by displaying such a library using DLL3, or at least a part, fragment, antigenic determinant or epitope thereof using one or more display techniques known in the field. Such libraries and techniques are, for example, described in documents WO 99 / 37681, WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694. Alternatively, semi-synthetic or enhanced synthetic libraries derived from virgin VHH libraries are used, such as VHH libraries obtained from virgin VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as, for example, described in document WO 00 / 43507.

[0187] In a further embodiment, yet another technique for obtaining VHH sequences directed against DLL3, involves adequately immunizing a transgenic mammal that has the capacity to express heavy chain antibodies (i.e., in order to increase an immune response and / or heavy chain antibodies). Petition 870260057806, dated 06 / 15 / 2026, page 91 / 374 81 / 256 heavy chain antibody-directed against DLL3), obtain a suitable biological sample from said transgenic mammal (such as a blood sample, serum sample or B cell sample) and then generate DLL3-directed VHH sequences, starting from said sample, using any suitable technique known in the field. For example, for this purpose, heavy chain antibody-expressing mice or rats and the additional methods and techniques described in WO 02 / 085945 and WO 04 / 049794 may be used.

[0188] In some embodiments, a single-domain anti-DLL3 antibody targeting DLL3 trispecific protein comprises a single-domain antibody with an amino acid sequence that corresponds 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 said naturally occurring VHH sequence (and in particular in the structure sequences) with one or more of the amino acid residues that occur at the corresponding position (or positions) in a VH domain of a conventional 4-chain antibody from a human (e.g., as indicated above). This can be accomplished in a manner known in the field, which will be clear to those skilled in the art, for example, based on the further description in this document.Again, it should be noted that such humanized single-domain anti-DLL3 antibodies of the development are obtained by any suitable manner known per se (i.e., as indicated under points (1)-(8) above) and thus are not strictly limited to polypeptides that were obtained using a polypeptide comprising a naturally occurring VHH domain as a starting material. In some additional embodiments, a single-domain anti-DLL3 antibody, as described herein, comprises a single-domain antibody with an amino acid sequence that corresponds to the sequence of. Petition 870260057806, dated 06 / 15 / 2026, page 92 / 374 82 / 256 amino acids from a naturally occurring VH domain that 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 occurring at the corresponding position(s) in a VHH domain of a heavy-chain antibody. Such camelization substitutions are preferably inserted at amino acid positions that form and / or are present at the VHVL interface and / or at so-called Camelidae marker residues (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence used as a starting material or starting point for generating or designing the camelized single domain is preferably a mammalian VH sequence, more preferably a human VH sequence, such as a VH3 sequence.However, it should be noted that such camelized anti-DLL3 single-domain antibodies of the breakthrough, in certain embodiments, are obtained by any suitable manner known in the field (i.e., as indicated under points (1)-(8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide comprising a naturally occurring VH domain as a starting material. For example, as further described herein, both humanization and camelization are performed by providing a nucleotide sequence encoding a naturally occurring VH domain or VH domain, respectively, and then by altering one or more codons in said nucleotide sequence such that the new nucleotide sequence encodes a humanized or camelized single-domain antibody, respectively.This nucleic acid can then be expressed to provide a desired single-domain anti-DLL3 antibody for development. Petition 870260057806, dated 06 / 15 / 2026, page 93 / 374 83 / 256 Alternatively, in other embodiments, based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, the amino acid sequence of the desired humanized or camelized single-domain anti-DLL3 antibody of the development, respectively, is designed and then synthesized de novo using known techniques for peptide synthesis. In some embodiments, based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, a nucleotide sequence encoding the desired humanized or camelized single-domain anti-DLL3 antibody of the development, respectively, is designed and then synthesized de novo using known techniques for nucleic acid synthesis, after which the nucleic acid thus obtained is expressed using known expression techniques, so as to provide the desired single-domain anti-DLL3 antibody of the development.

[0189] Other suitable methods and techniques for obtaining the single-domain anti-DLL3 antibody from development and / or nucleic acids encoding the same, starting from naturally occurring VHH sequences or VHH sequences, for example, comprise combining one or more parts of one or more naturally occurring VHH sequences (such as one or more frame (FR) sequences and / or complementarity-determining region (CDR) sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences) and / or one or more synthetic or semi-synthetic sequences, in a suitable manner, so as to provide a single-domain anti-DLL3 antibody from development or a nucleotide or nucleic acid sequence encoding the same.

[0190] In some embodiments, the DLL3-binding domain is a specific anti-DLL3 antibody comprising a chain-variable CDR1 complementarity-determining region. Petition 870260057806, dated 06 / 15 / 2026, page 94 / 374 84 / 256 heavy, one variable CDR2 heavy chain, one variable CDR3 heavy chain, one variable CDR1 light chain, one variable CDR2 light chain and one variable CDR3 light chain. In some embodiments, the DLL3-binding domain comprises any DLL3-binding domain which includes, but is not limited to, domains of a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or antigen-binding fragments such as single-domain antibodies (sdAb), Fab, Fab', F(ab)2 and Fv fragments, fragments comprising one or more CDRs, single-chain antibodies (e.g., single-chain Fv fragments (scFv)), disulfide-stabilized Fv fragments (dsFv), heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain dimers and monomers, light chain dimers and monomers, and dimers consisting of a heavy chain and a light chain.In some embodiments, the DLL3-binding domain is a single-domain antibody. In some embodiments, the anti-DLL3 single-domain antibody comprises complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3 heavy chain variables.

[0191] In some embodiments, the DLL3-binding domain is a polypeptide comprising an amino acid sequence that is comprised of four structural regions / sequences (f1f4) interrupted by three complementarity-determining regions / sequences, as represented by the formula: f1-r1-f2-r2f3-r3-f4, wherein r1, r2, and r3 are complementarity-determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are structural residues. The structural residues of the DLL3-binding protein of the present disclosure comprise, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 or 94 amino acid residues, and the complementarity-determining regions comprise, for example, 24, Petition 870260057806, dated 06 / 15 / 2026, page 95 / 374 85 / 256 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues. In some embodiments, the DLL3 binding domain comprises an amino acid sequence selected from SEQ ID Nos. 1-442 and 1886. In some embodiments, CDR1 of the DLL3 binding domain comprises a sequence selected from SEQ ID Nos. 443-884 and 1887, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 443-884 and 1887. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID Nos. 885-1326 and 1888, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID Nos. 8851326 and 1888. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID Nos. 1327-1768 and 1889, or one or more substitutions relating to a sequence selected from SEQ ID Nos.1327-1768 and 1889.

[0192] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID Nos. 443-884 and 1887 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid selected from SEQ ID Nos. 443-884 and 1887. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID Nos. 885-1326 and 1888 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid sequence selected from SEQ ID Nos. 885-1326 and 1888. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID Nos. 1327-1768 and 1889 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in a selected sequence of SEQ ID Nos. 1327-1768 and 1889.

[0193] In some modalities, CDR1 comprises a Petition 870260057806, dated 06 / 15 / 2026, p. 96 / 374 86 / 256 amino acid sequence selected from SEQ ID Nos. 495-528 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid selected from SEQ ID Nos. 495-528. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID Nos. 937-970 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid sequence selected from SEQ ID Nos. 937-970. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID Nos. 1379-1412 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in a sequence selected from SEQ ID Nos. 1379-1412.

[0194] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID Nos. 529-809 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid selected from SEQ ID Nos. 529-809. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID Nos. 971 to 1251 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid sequence selected from SEQ ID Nos. 971 to 1251. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID Nos. 1379 to 1412 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in a sequence selected from SEQ ID Nos. 1379-1412.

[0195] In some embodiments, CDR1 comprises an amino acid sequence selected from SEQ ID Nos. 810-884 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in a Petition 870260057806, dated 06 / 15 / 2026, p. 97 / 374 87 / 256 amino acid selected from SEQ ID Nos. 810-884. In some embodiments, CDR2 comprises an amino acid sequence selected from SEQ ID Nos. 1252 to 1326 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in an amino acid sequence selected from SEQ ID Nos. 1252 to 1326. In some embodiments, CDR3 comprises an amino acid sequence selected from SEQ ID Nos. 1692 to 1768 or a variant with one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions in a sequence selected from SEQ ID Nos. 1692 to 1768.

[0196] In various embodiments, the DLL3 binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID Nos. 1-442 and 1886.In various embodiments, the DLL3-binding domain of the present disclosure is at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID Nos. 53-86.

[0197] 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 Petition 870260057806, dated 06 / 15 / 2026, p. 98 / 374 88 / 256%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical to an amino acid sequence selected from SEQ ID Nos. 87-367.

[0198] 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.

[0199] 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.

[0200] In some embodiments, the DLL3-binding domain of the trispecific DLL3-targeting protein has transreactivity with human and cynomolgus DLL3. In some embodiments, the DLL3-binding domain is specific for human DLL3. In certain embodiments, the DLL3-binding domain disclosed herein binds to human DLL3 with a human Kd (hKd). In certain embodiments, the DLL3-binding domain disclosed herein binds to cynomolgus DLL3 with a cynomolgus Kd (cKd). In certain embodiments, the DLL3-binding domain disclosed herein binds to both Petition 870260057806, dated 06 / 15 / 2026, p. 99 / 374 89 / 256 to cynomolgus DLL3 as to 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., hKd and cKd values ​​do not differ by more than ± 10%). In some embodiments, the hKd and cKd range is from about 0.001 nM to about 500 nM. In some embodiments, the hKd and cKd range is from about 0.001 nM to about 450 nM. In some embodiments, the hKd and cKd range is from about 0.001 nM to about 400 nM. In some modes, the hKd range and the cKd range from about 0.001 nM to about 350 nM. In some modes, the hKd range and the cKd range from about 0.001 nM to about 300 nM. In some modes, the hKd range and the cKd range from about 0.001 nM to about 250 nM. In some modes, the hKd range and the cKd range from about 0.001 nM to about 200 nM. In some modes, the hKd range and the cKd range from about 0.001 nM to about 150 nM. In some modes, the hKd range and the cKd range from about 0.001 nM to about 100 nM. In some modes, the hKd range and the cKd range from approximately 0.1 nM to approximately 90 nM. In some modes, the hKd range and the cKd range from approximately 0.2 nM to approximately 80 nM. In some modes, the hKd range and the cKd range from approximately 0.3 nM to approximately 70 nM. In some modes, the hKd range and the cKd range from approximately 0.4 nM to approximately 50 nM. In some modes, the hKd range and the cKd range from approximately 0.5 nM to approximately 30 nM. In some modes, the hKd range and the cKd range from approximately 0.6 nM to approximately 10 nM. In some modes, the hKd range and the cKd range are from approximately 0.7 nM to approximately 8 nM. In some modes, the hKd range and the cKd range are from approximately 0.8 nM to approximately 6 nM. In some modes, the hKd range and the cKd range are from approximately 0.9 nM to approximately 4 nM. In some modes, the hKd range and the cKd range are from approximately 1 nM to approximately 2 nM. Petition 870260057806, dated 06 / 15 / 2026, p. 100 / 374 90 / 256

[0201] In certain embodiments, the DLL3-binding domains of the present disclosure preferentially bind membrane-bound DLL3 over soluble DLL3. Membrane-bound DLL3 refers to the presence of DLL3 on or at the cell membrane surface of a cell expressing DLL3. Soluble DLL3 refers to DLL3 that is no longer on or at the cell membrane surface of a cell that expresses or has expressed DLL3. In certain cases, soluble DLL3 is present in the blood and / or lymphatic circulation in an individual. In one embodiment, the DLL3-binding proteins bind membrane-bound DLL3 at least 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, 100 times, 500 times, or 1000 times more than soluble DLL3. In one embodiment, the antigen-binding proteins of the present disclosure preferentially bind to membrane-bound DLL3 30 times more than to soluble DLL3.The determination of the preferential binding of an antigen-binding protein to membrane-bound DLL3 via soluble DLL3 can be readily determined using well-known assays in the art.

[0202] In some embodiments, any of the previously mentioned DLL3-binding domains (e.g., single-domain anti-DLL3 antibodies of SEQ ID Nos. 1-442 and 1886) are affinity peptide-tagged for ease of purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also referred to as 6X-his (SEQ ID No. 1819).

[0203] In some embodiments, any of the previously mentioned DLL3-binding domains (e.g., single-domain anti-DLL3 antibodies of SEQ ID Nos. 1-442 and 1886) are affinity peptide-tagged for ease of purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also referred to as 6X-his (SEQ ID No. 1819). Integration into chimeric antigen receptors (CARs) Petition 870260057806, dated 06 / 15 / 2026, page 101 / 374 91 / 256

[0204] The trispecific antigen-binding proteins targeting DLL3 of the present disclosure can, in certain examples, be incorporated into a chimeric antigen receptor (CAR). A genetically modified immune effector cell, a T cell or NK cell, can be used to express a CAR that includes a trispecific anti-DLL3-targeting protein containing a single-domain anti-DLL3 antibody, as described herein. In one embodiment, the CAR that includes a trispecific anti-DLL3-targeting protein as described herein is connected to a transmembrane domain via a hinge region, and, additionally, a costimulatory domain, a functional signaling domain obtained from OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) or 4-1BB. In some forms, the CAR additionally comprises a sequence that encodes an intracellular signaling domain, such as 4-1BB and / or CD3 zeta. Tumor growth-reducing properties

[0205] In certain embodiments, trispecific proteins targeting DLL3-revealing tumor cells reduce the growth of tumor cells in vivo when administered to an individual who has tumor cells expressing DLL3. The measurement of tumor cell growth reduction can be determined by several different methodologies well known in the art. Non-limiting examples include direct measurement of tumor size, measurement of excised tumor mass and comparison to control individuals, measurement by means of imaging techniques (e.g., CT or MRI) that may or may not use isotopes or luminescent molecules (e.g., luciferase) to enhance analysis, and the like.

[0206] In specific modalities, administration of trispecific development proteins results in a reduction of tumor cell growth in vivo compared to a control antigen-binding agent by at least about 10%, Petition 870260057806, dated 06 / 15 / 2026, page 102 / 374 92 / 256%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, where approximately 100% reduction in tumor growth indicates complete response and tumor disappearance. In additional modalities, administration of trispecific development proteins results in a reduction of tumor cell growth in vivo compared to a control antigen-binding agent by approximately 50-100%, approximately 75-100%, or approximately 90-100%. In additional embodiments, administration of trispecific development proteins results in a reduction of tumor cell growth in vivo compared to a control antigen-binding agent by approximately 50-60%, approximately 60-70%, approximately 70-80%, approximately 80-90%, or approximately 90-100%. Trispecific Protein Modifications that Target DLL3

[0207] The trispecific proteins targeting DLL3 described in this document encompass derivatives or analogues in which (i) an amino acid is replaced with an amino acid residue that is not encoded by the genetic code, (ii) the mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence or a sequence for protein purification.

[0208] 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, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent transreactivities, cystine formation, pyroglutamate formation, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, Petition 870260057806, dated 06 / 15 / 2026, page 103 / 374 93 / 256 phosphorylation, prenylation, racemization, selenoylation, sulfation, RNA transfer-mediated addition of amino acids to proteins such as arginylation and ubiquitination.

[0209] Modifications are produced anywhere in the trispecific proteins targeting DLL3 described herein, including the main peptide structure, amino acid side chains, and amino or carboxyl termini. Certain common peptide modifications that are useful for modifying trispecific proteins targeting DLL3 include glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, blocking of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, and ADP ribosylation.

[0210] In some embodiments, a derivative of the trispecific protein targeting DLL3, as described herein, comprises immunoreactive modulator derivatives and antigen-binding molecules comprising one or more modifications.

[0211] In some embodiments, the three-specific DLL3-binding molecules of the development are monovalent or multivalent, bivalent, trivalent, etc. As used in this document, the term valence refers to the number of potential target binding sites associated with an antibody. Each target binding site binds specifically to a target molecule or a specific position or site on a target molecule. When an antibody is monovalent, each binding site on the molecule will bind specifically to a single antigen position or epitope. When an antibody comprises more than one target binding site (multivalent), each target binding site may bind specifically to the same molecules or different molecules (e.g., it may bind to different ligands or different antigens, or different epitopes or positions on the same antigen). Petition 870260057806, dated 06 / 15 / 2026, p. 104 / 374 94 / 256

[0212] In some embodiments, the DLL3-targeting trispecific proteins of this disclosure contain, among others, one or more additional amino acid residue substitutions, mutations, and / or modifications that result in a compound with preferred characteristics including, but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, reduced immunogenicity, increased production, altered Fc ligand binding to an Fc receptor (FcR), enhanced or reduced ADCC (antibody-dependent cell-mediated cytotoxicity) or CDC (complement-dependent cytotoxicity) activity, altered glycosylation and / or disulfide bonds, and modified binding specificity. In some cases, these DLL3-targeting trispecific protein variants are advantageously used to enhance the effective anti-neoplastic properties of the disclosed DLL3-targeting trispecific proteins.

[0213] In some embodiments, the trispecific proteins targeting DLL3 have half-lives in mammals, such as in a human, or in a cynomolgus monkey, less than about 5 days, about 5 days, greater than about 5 days, greater than 10 days, greater than about 15 days, greater than about 20 days, greater than about 25 days, greater than about 30 days, greater than about 35 days, greater than about 40 days, greater than about 45 days, greater than about 2 months, greater than about 3 months, greater than about 4 months, or greater than about 5 months. The increased half-life, in some cases, results in a higher serum titer which thereby reduces the frequency of administration of the trispecific proteins targeting DLL3, reduces the concentration of antibodies to be administered, or both.

[0214] Still other embodiments comprise one or more genetically modified glycoforms, that is, a trispecific binding protein targeting DLL3 which comprises a pattern Petition 870260057806, dated 06 / 15 / 2026, p. 105 / 374 95 / 256 altered glycosylation or altered carbohydrate composition that is covalently attached to the protein. Genetically modified glycoforms are useful, in some cases, for a variety of purposes, including, but not limited to, improving or reducing effector function, increasing the affinity of the trispecific protein for a target, or facilitating the production of the trispecific protein. In certain embodiments where reduced effector function is desired, the molecule is genetically modified to express an aglycosylated form. Substitutions that result in the elimination of one or more variable-region structure glycosylation sites, thus eliminating glycosylation at such a site, are included in some embodiments. On the other hand, improved effector functions or enhanced binding are provided to the Fc containing trispecific proteins of this development by genetic modification at one or more additional glycosylation sites, in some cases.

[0215] The trispecific proteins that target DLL3 are, in some cases, differentially modified during or after production by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding to an antibody molecule or other cellular ligand, etc. Any of the various chemical modifications are performed by techniques including, but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

[0216] Various post-translational modifications also covered by include, for example, N-linked and O-linked carbohydrate chains, N-terminal or C-terminal end processing, attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked and O-linked carbohydrate chains, and addition or deletion of a Petition 870260057806, dated 06 / 15 / 2026, p. 106 / 374 96 / 256 N-terminal methionine residue as a result of prokaryotic host cell expression. Furthermore, trispecific binding proteins targeting DLL3 are, in some cases, modified with a detectable label, such as an enzymatic, fluorescent, radioisotopic, or affinity label, to allow detection and isolation of the modulator. Polynucleotides encoding trispecific proteins targeting DLL3

[0217] Also provided in some embodiments are polynucleotide molecules encoding a trispecific anti-DLL3 binding protein described in this document. In some embodiments, the polynucleotide molecules are provided as a DNA construct. In other embodiments, the polynucleotide molecules are provided as a messenger RNA transcript.

[0218] Polynucleotide molecules are constructed by methods known as combining genes encoding the three binding domains separated by peptide linkers or, in other embodiments, directly linked by a peptide linker, into a simple genetic construct operationally linked to a suitable promoter, and optionally a suitable transcription terminator, and which expresses the same in bacteria or another suitable expression system such as, for example, CHO cells. In embodiments where the DLL3-binding domain is a small molecule, the polynucleotides contain genes encoding the CD3-binding domain and the half-life extension domain. In embodiments where the half-life extension domain is a small molecule, the polynucleotides contain genes encoding the CD3-binding and DLL3-binding domains.Depending on the vector and host system used, any variety of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. The promoter is selected so that it triggers the expression of the... Petition 870260057806, dated 06 / 15 / 2026, page 107 / 374 97 / 256 polynucleotide in the respective host cell.

[0219] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents an additional embodiment. This recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagomides, phage derivatives, viruses (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, lentiviruses, and the like), and cosmids.

[0220] A variety of vector / host expression systems can be used to contain and express the polynucleotide encoding the described trispecific antigen-binding protein polypeptide. Examples of expression vectors for expression in E. coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0221] Thus, trispecific proteins targeting DLL3, as described in this document, in some embodiments, are produced by introducing a vector encoding the protein as described above into a host cell and culturing said host cell under conditions in which the protein domains are expressed, can be further isolated and optionally purified. Pharmaceutical Compositions

[0222] Pharmaceutical compositions comprising a trispecific anti-DLL3 binding protein described herein, a vector comprising the polynucleotide encoding the polypeptide of the trispecific proteins targeting DLL3 or a host cell transformed by such vector, and at least one pharmaceutically acceptable carrier are also provided in some embodiments. The term pharmaceutically acceptable carrier includes, but is not limited to, any carrier that does not interfere with effectiveness. Petition 870260057806, dated 06 / 15 / 2026, page 108 / 374 98 / 256 of the biological activity of the ingredients and that it is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Such carriers can be formulated by conventional methods and can be administered to the individual in an appropriate dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservatives, emulsifying agents, and dispersing agents. Prevention of microorganism action can be ensured by the inclusion of various antibacterial and antifungal agents. An additional embodiment provides one or more of the trispecific proteins that target DLL3 described above packaged in lyophilized form, or packaged in an aqueous medium.

[0223] In some embodiments of the pharmaceutical compositions, the DLL3-targeting trispecific proteins described herein are encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposomes, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical compositions, the DLL3-targeting trispecific protein is attached to liposomes. In some examples, the DLL3-targeting trispecific proteins are conjugated to the surface of liposomes. In some examples, the DLL3 trispecific antigen-binding protein is encapsulated in the shell of a liposome. In some cases, the liposome is a cationic liposome.

[0224] The trispecific proteins that target DLL3 described in this document are contemplated for use as a medicine. Administration is effective in different ways, by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some modalities, Petition 870260057806, dated 06 / 15 / 2026, p. 109 / 374 99 / 256 The route of administration depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any patient depend on many factors, including patient size, body surface area, age, sex, the particular compound to be administered, time and route of administration, type of therapy, general health, and other drugs that are administered concomitantly. An effective dose refers to amounts of the active ingredient that are sufficient to affect the course and severity of the disease, leading to the reduction or remission of such pathology, and can be determined using known methods.

[0225] In some embodiments, the trispecific proteins targeting DLL3 of this development are administered at a dosage of up to 10 mg / kg at a frequency of once a week. In some cases, the dosage is in the range of about 1 ng / kg to about 10 mg / kg. In some modalities, the dose is approximately 1 ng / kg to approximately 10 ng / kg, approximately 5 ng / kg to approximately 15 ng / kg, approximately 12 ng / kg to approximately 20 ng / kg, approximately 18 ng / kg to approximately 30 ng / kg, approximately 25 ng / kg to approximately 50 ng / kg, approximately 35 ng / kg to approximately 60 ng / kg, approximately 45 ng / kg to approximately 70 ng / kg, approximately 65 ng / kg to approximately 85 ng / kg, approximately 80 ng / kg to approximately 1 pg / kg, approximately 0.5 pg / kg to approximately 5 pg / kg, approximately 2 pg / kg to approximately 10 pg / kg, approximately 7 pg / kg to approximately 15 pg / kg, approximately 12 pg / kg to approximately 25 pg / kg, approximately 20 pg / kg to approximately 50 pg / kg, approximately 35 pg / kg to approximately 70 pg / kg, approximately 45 pg / kg to approximately 80 pg / kg, approximately 65 pg / kg to approximately 90 pg / kg, approximately 85 pg / kg to approximately 0.1 mg / kg, approximately 0.095 mg / kg to approximately 10 mg / kg. In some cases, the dosage is approximately 0.1 mg / kg to approximately 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. Petition 870260057806, dated 06 / 15 / 2026, page 110 / 374 Dosages vary from approximately 100 / 256 mg / kg, approximately 4.5 mg / kg to approximately 6 mg / kg, approximately 5.5 mg / kg to approximately 7 mg / kg, approximately 6.5 mg / kg to approximately 8 mg / kg, approximately 7.5 mg / kg to approximately 9 mg / kg, or approximately 8.5 mg / kg to approximately 10 mg / kg. The frequency of administration, in some forms, is approximately less than daily, every other day, less than once daily, twice weekly, weekly, 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 frequency of administration is weekly. In some cases, the frequency of administration is weekly and the dosage is up to 10 mg / kg. In some cases, the duration of administration is from approximately 1 day to approximately 4 weeks or more. Treatment methods

[0226] In some embodiments, DLL3-binding proteins, or trispecific proteins targeting DLL3 of the present disclosure are administered to treat a neoplastic condition.Neoplastic conditions, in some forms, are benign or malignant; solid tumors or other blood neoplasms; and, in some modalities, are selected from the group that includes, but is not limited to: adrenal gland tumors, AIDS-associated cancers, alveolar soft tissue sarcoma, astrocytic tumors, autonomic ganglion tumors, bladder cancer (squamous cell carcinoma and transitional cell carcinoma), blastocoelic disorders, bone cancer (adamantinoma, bone aneurysm cysts, osteochondroma, osteosarcoma), spinal cord and brain cancers, metastatic brain tumors, breast cancer including triple-negative breast cancer, carotid body tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign cutaneous fibrous histiocytomas, desmoplastic small round cell tumors, ependymomas, disorders. Petition 870260057806, dated 06 / 15 / 2026, p. 111 / 374 101 / 256 epithelial, Ewing's tumors, extraskeletal myxpoid chondrosarcoma, osteogenesis imperfecta, fibrous dysplasia of bone, gallbladder and bile duct cancers, gastric cancer, gastrointestinal cancer, gestational trophoblastic disease, germ cell tumors, glandular disorders, head and neck cancers, hypothalamic cancer, bowel cancer, islet cell tumors, Kaposi's sarcoma, renal cancer (nephroblastoma, papillary renal cell carcinoma), leukemias, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, liver cancer (hepatoblastoma, hepatocellular carcinoma), lymphomas, lung cancers (small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc.).), macrophage disorders, medulloblastoma, melanoma, meningiomas, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancers, papillary thyroid carcinomas, parathyroid tumors, pediatric cancers, peripheral nerve sheath tumors, pheochromocytoma, pituitary tumors, prostate cancer, posterior ocular melanoma, rare hematological disorders, metastatic renal cancer, rhabdoid tumor, rhabdomyosarcoma, sarcomas, skin cancer, soft tissue sarcomas, squamous cell carcinoma, stomach cancer, stromal disorders, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancers (cervical carcinoma, endometrial carcinoma, and leiomyoma).

[0227] In certain embodiments, DLL3-binding proteins, or trispecific DLL3-targeting proteins of the present disclosure, are used as a first-line therapy and administered to individuals who have not previously been treated for the cancerous condition. In other embodiments, trispecific DLL3-targeting proteins of the present disclosure Petition 870260057806, dated 06 / 15 / 2026, page 112 / 374 102 / 256 are used to treat individuals who have been previously treated (with a DLL3-targeting trispecific protein of this development or with another anticancer agent) and have experienced recurrence or have been determined to be refractory to prior treatment. In some modalities, DLL3-targeting trispecific proteins of this development are used to treat individuals who have recurrent tumors.

[0228] In some respects, DLL3-binding proteins, or DLL3-targeting trispecific proteins of the present disclosure, are administered to treat a proliferative disorder comprising a solid tumor which includes, but is not limited to, adrenal, liver, kidney, bladder, breast, gastric, ovarian, cervical, uterine, esophageal, colorectal, prostate, pancreatic, lung (both small cell and non-small cell), thyroid, carcinomas, sarcomas, glioblastomas and various head and neck tumors.

[0229] In some embodiments, DLL3-binding proteins, or trispecific DLL3-targeting proteins of the present disclosure, are administered to an individual suffering from melanoma. In some embodiments, trispecific DLL3-targeting proteins of the present disclosure are used to diagnose, monitor, treat, or prevent melanoma.The term melanoma, as used in this document, includes all types of melanoma, including, but not limited to, primary melanoma, malignant melanoma, cutaneous melanoma, extracutaneous melanoma, superficial spreading melanoma, polypoid melanoma, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, nodular malignant melanoma, lentigo maligna melanoma, lentiginous melanoma, malignant lentiginous melanoma, mucosal lentiginous melanoma, mucosal melanoma, acral lentiginous melanoma, soft tissue melanoma, ocular melanoma, invasive melanoma, familial atypical melanoma and nevus syndrome (FAM-M), desmoplastic malignant melanoma, or ocular melanoma. Petition 870260057806, dated 06 / 15 / 2026, page 113 / 374 103 / 256

[0230] 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. Thus, in some modalities where DLL3-binding proteins, or trispecific proteins that target DLL3 from development, are incorporated into a chimeric antigen receptor expressed on lymphocytes, the resulting “DLL3-sensitized lymphocytes” (e.g., natural killer cells or T cells that immunospecifically recognize a DLL3 determinant) have the ability to effectively mount an immune response directed at abnormal DLL3-positive cells, including cancer stem cells. This ability to effectively eliminate tumorigenic seed cells is often critical in reducing the possibility of tumor recurrence or metastasis.In some modalities, these DLL3-sensitized lymphocytes are used in combination with other therapeutic agents or as part of a maintenance regimen that follows standard care treatments.

[0231] More generally, a chimeric antigen receptor is an artificially constructed hybrid protein or polypeptide that contains or comprises an antigen-binding domain of an antibody linked to a signaling domain (e.g., T cell signaling or T cell activation domains). In some embodiments, CARs comprising the presently disclosed DLL3-targeting trispecific binding protein have the ability to redirect the specificity and reactivity of sensitized lymphocytes (e.g., T cells) to DLL3-positive target cells in an MHC-unrestricted manner by exploiting the antigen-binding properties of antibodies or antigen-binding fragments thereof. MHC-unrestricted antigen recognition provides T cell-expressing DLL3 CARs with the ability to recognize tumorigenic DLL3 independently of processing. Petition 870260057806, dated 06 / 15 / 2026, page 114 / 374 104 / 256 antigen, thus bypassing a major tumor escape mechanism. Furthermore, when expressed on T cells, CARs advantageously do not dimerize with alpha and beta chains of endogenous T cell receptors (TCRs).

[0232] In selected aspects, DLL3-binding proteins, or DLL3-targeting trispecific proteins, are incorporated into a chimeric antigen receptor (CAR) and the DLL3 CAR is delivered in an effective CAR-based therapy for the treatment of lung cancer, including the following subtypes: small cell lung cancer, non-small cell lung cancer (e.g., squamous non-small cell lung cancer or squamous small cell lung cancer), and large cell neuroendocrine carcinoma (LCNEC).

[0233] In some embodiments, DLL3-binding proteins, or DLL3-sensitive lymphocytes, are administered to patients exhibiting limited-stage or extensive-stage disease. In other embodiments, revealed DLL3-targeting trispecific antibodies are administered to refractory patients (i.e., those whose disease recurs during or shortly after completion of an initial course of therapy); sensitive patients (i.e., those whose recurrence is longer than 23 months after primary therapy); or patients exhibiting resistance to a platinum-based agent (e.g., carboplatin, cisplatin, oxaliplatin) and / or a taxane (e.g., docetaxel, paclitaxel, larotaxel, or cabazitaxel). In another embodiment, revealed DLL3 CAR treatments are effective in treating ovarian cancer, including serous ovarian carcinoma and papillary serous ovarian carcinoma.

[0234] The revealed DLL3-binding proteins, or trispecific DLL3-targeting binding proteins, in some embodiments, are used to prevent, treat, or diagnose tumors with neuroendocrine features or phenotypes that Petition 870260057806, dated 06 / 15 / 2026, p. 115 / 374 105 / 256 include neuroendocrine tumors. True or canonical neuroendocrine tumors (NETs) arising from the scattered endocrine system are relatively rare, with an incidence of 2-5 per 100,000 people, but highly aggressive. Neuroendocrine tumors occur in the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid cancer), and lung (small cell lung carcinoma and large cell neuroendocrine carcinoma). These tumors can secrete various hormones, including serotonin and / or chromogranin A, which can cause debilitating symptoms known as carcinoid syndrome. Such tumors can be identified by positive immunohistochemical markers such as neuron-specific enolase (NSE, also known as enolase gamma, gene symbol=ENO2), CD56 (or NCAM1), chromogranin A (CHGA), and synaptophysin (SYP), or by genes known to exhibit elevated expression such as ASCL1.Traditional chemotherapies have not been particularly effective in treating neuroendocrine tumors, and liver metastasis is a common outcome. In some modalities, trispecific antibodies targeting DLL3 have been advantageously used to treat neuroendocrine tumors, and in some modalities, the same antibodies are used to treat, prevent, or diagnose neuroendocrine pseudotumors (pNETs) that genotypically or phenotypically mimic, resemble, or exhibit common features with canonical neuroendocrine tumors. Neuroendocrine pseudotumors, or tumors with neuroendocrine features, are tumors that arise from cells of the diffuse neuroendocrine system or from cells in which a neuroendocrine differentiation cascade has been abnormally reactive during the oncogenic process. Such pNETs commonly share certain phenotypic or biochemical characteristics with traditionally defined neuroendocrine tumors, including... Petition 870260057806, dated 06 / 15 / 2026, p. 116 / 374 106 / 256 ability to produce subsets of biologically active amines, neurotransmitters, and peptide hormones. Histologically, such tumors (NETs and pNETs) share a common appearance in that they frequently show small, densely connected cells with minimal cytoplasm of mild cytopathology and round to oval stippled nuclei. In some embodiments of the present discovery, commonly expressed histological or genetic markers that are used to define neuroendocrine tumors and neuroendocrine pseudotumors include, but are not limited to, chromogranin A, CD56, synaptophysin, PGP9.5, ASCL1, and neuron-specific enolase (NSE).Consequently, in some embodiments, the DLL3-targeting trispecific protein of the present development, DLL3 CAR, or DLL3-sensitized lymphocytes, or any combination thereof, are beneficially used to treat both neuroendocrine pseudotumors and canonical neuroendocrine tumors, such as to treat neuroendocrine tumors (both NETs and pNETs) arising in the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid cancer), and lung (small cell lung carcinoma and large cell neuroendocrine carcinoma). Furthermore, in some modalities, the DLL3 trispecific targeting protein, the DLL3 CAR, or DLL3-sensitized lymphocytes, or any combination thereof, are used to treat tumors that express one or more markers such as NSE, CD56, synaptophysin, chromogranin A, ASCL1, or PGP9.5 (UCHL1).In some modalities, the DLL3 trispecific protein that targets the development, the DLL3 CAR, or DLL3-sensitized lymphocytes, or any combination thereof, are used to treat an individual suffering from a tumor that is NSE+ or CD56+ or PGP9.5+ or ASCL1+ or SYP+ or CHGA+ or any combination thereof. Petition 870260057806, dated 06 / 15 / 2026, p. 117 / 374 107 / 256

[0235] In another embodiment, DLL3 trispecific targeting protein, DLL3 CAR, or DLL3 sensitized lymphocytes, or any combination thereof, are used in maintenance therapy to reduce or eliminate the chance of tumor recurrence after initial disease presentation. In some cases, the disorder has been treated and the initial tumor mass eliminated, reduced, or otherwise improved, so that the patient is asymptomatic or in remission. At this point, the individual is administered pharmaceutically effective amounts of the DLL3-binding proteins revealed, DLL3 trispecific targeting protein, DLL3 CAR, or DLL3 sensitized lymphocytes, or any combination thereof, one or more times regardless of whether there is little or no indication of disease using standard diagnostic procedures.In some modalities, the DLL3 trispecific protein that targets the developing cell, DLL3 CAR, or DLL3-sensitized lymphocytes, or any combination thereof, are administered on a regular schedule over a period of time, such as weekly, every two weeks, monthly, every six weeks, every two months, every three months, every six months, or annually, for example, to reduce the potential for disease recurrence. Furthermore, such treatments are, in some modalities, continued for a period of weeks, months, years, or even indefinitely, depending on the patient's response and clinical and diagnostic parameters.

[0236] In yet another embodiment, DLL3-binding proteins, DLL3 trispecific targeting protein, DLL3 CAR, or DLL3-sensitized lymphocytes, or any combination thereof, are used to prophylactically or as adjuvant therapy prevent or reduce the possibility of tumor metastasis after a treatment procedure. As used in a present disclosure, a “treatment procedure is Petition 870260057806, dated 06 / 15 / 2026, p. 118 / 374 108 / 256 is broadly defined and means any procedure, technique, or method that eliminates, reduces, treats, or improves a tumor or tumor proliferation. Exemplary treatment procedures include, but are not limited to, surgery, radiation treatments (i.e., beam radiation), chemotherapy, immunotherapy, or ablation. In some modalities, at appropriate times, DLL3-binding proteins, DLL3 trispecific targeting protein, DLL3 CAR, or DLL3-sensitized lymphocytes, or any combination thereof, are administered as suggested by clinical, diagnostic, or theranostic procedures to reduce tumor metastasis. In some modalities, the dosing regimen is accompanied by appropriate diagnostic or monitoring techniques that allow it to be modified.

[0237] Still other modalities of detection comprise administering DLL3-binding proteins, DLL3 trispecific targeting protein, DLL3 CAR, or DLL3-sensitized lymphocytes, or any combination thereof, to individuals who are asymptomatic but are at risk of developing a proliferative disorder. That is, in some modalities, DLL3-binding proteins, DLL3 trispecific targeting protein, DLL3 CAR, or DLL3-sensitized lymphocytes, or any combination thereof, are used preventively and given to patients who have been screened or tested and have one or more observed risk factors (e.g., genomic indications, family history, in vivo or in vitro test results, etc.) but have not developed neoplasia.In such cases, those skilled in the art would have the ability to determine an effective dosage regimen through empirical observation or through acceptable clinical practices.

[0238] As used in this document, in some forms, treatment or to treat or treated refers to Petition 870260057806, dated 06 / 15 / 2026, page 119 / 374 109 / 256 Therapeutic treatment where the objective is to slow down (reduce) an undesirable physiological condition, disorder, or disease, or to obtain beneficial or desired clinical outcomes. For the purposes described herein, beneficial or desired clinical outcomes include, but are not limited to, symptom relief; reduction in the extent of the condition, disorder, or disease; stabilization (i.e., no worsening) of the condition, disorder, or disease state; delay in the onset or reduction in the progression of the condition, disorder, or disease; relief of the condition, disorder, or disease state; and remission (whether partial or total), whether detectable or undetectable, or potentiation or improvement of the condition, disorder, or disease. Treatment includes inducing a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to expected survival if no treatment is received.In other modalities, treatment or treating or treated refers to prophylactic measures, where the objective is to delay the onset of or reduce the severity of an unwanted physiological condition, disorder, or disease, such as, for example, a person who is predisposed to a disease (for example, an individual who carries a genetic marker for a disease such as breast cancer).

[0239] In some embodiments of the methods described herein, DLL3-binding proteins, trispecific DLL3-targeting proteins, or compositions, as described herein, are administered in combination with an agent for the treatment of a particular disease, disorder, or condition. Agents include, but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroidal, peptide, and the like), radiotherapies (gamma rays, X-rays, and / or targeted delivery of radioisotopes, microwaves, UV radiation, and the like), gene therapies (e.g., antisense, retroviral therapy, and...). Petition 870260057806, dated 06 / 15 / 2026, page 120 / 374 110 / 256 similar) and other immunotherapies. In some embodiments, an anti-DLL3 binding protein, or a trispecific anti-DLL3 targeting protein, as described herein, is administered in combination with anti-diarrheal agents, anti-emetic agents, analgesics, opioids, and / or nonsteroidal anti-inflammatory agents. In some embodiments, an anti-DLL3 binding protein, or a trispecific anti-DLL3 targeting protein, as described herein, is administered in combination with anticancer agents. Non-limiting examples of anticancer agents that may be used in the various embodiments of the disclosure, including pharmaceutical compositions and disclosure dosage forms and kits, include: acivicin; aclarubicin; acodazol hydrochloride; acronin; adozelesin; aldesleucin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; ansaccharin; anastrozole; anthramycin; asparaginase; asperline; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisanthrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucine; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; phosphate; Petition 870260057806, dated 06 / 15 / 2026, p. 121 / 374 111 / 256 fludarabine; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexole sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitochromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxysuran; paclitaxel; pegaspargase; peliomycin; pentamustine;peplomycin sulfate; perfosfamide; pipobromane; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparphosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogallan sodium; tegafur; teloxantrone hydrochloride; temoporphine; teniposide; teroxirone; testolactone; thiamipine; thioguanine; thiotepa; thiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglicinate sulfate; Petition 870260057806, dated 06 / 15 / 2026, p. 122 / 374 112 / 256 vinleurosin sulfate; vinorelbine tartrate; vinzolidine sulfate; vinzolidine sulfate; vorozol; zeniplatin; zinostatin; zorubicin hydrochloride. Other examples of anticancer drugs include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethinyluracil; abiraterone; aclarubicin; acilfulvene; adecipenol; adozelesin; aldesleucine; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; ansaccharin; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; D antagonist; G antagonist; antarelix; anti-dorsalizing morphogenetic protein 1; antiandrogen, prostate carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin;azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorines; benzoylstaurosporine; beta-lactam derivatives; beta-alethin; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisanthrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; butyonin sulfoximine; calcipotriol; calfostine C; camptothecin derivatives; canaripox IL-2; capecitabine; carboxamide-aminotriazole; carboxiamidotriazole; CaRest M3; CARN 700; cartilage-derived inhibitor; carzelesin; casein kinase inhibitors (CKIs); castanospermine; cecropin B; cetrorelix; chlorines; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomiphene analogues; clotrimazole; colismycin A; colimycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; chrysnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentantraquinones; cycloplatam; Petition 870260057806, dated 06 / 15 / 2026, p. 123 / 374 113 / 256 cypemycin; cytarabine ocphosphate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodimmine B; deslorelin; dexamethasone; dexiphosphamide; dexrazoxane; dexverapamil; diaziquone; didemmine B; didox; diethylnorspermine; dihydro-5-azacitidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxyfluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorucin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texafirin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin;hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; hydramantoin; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulating peptides; insulin-like growth factor I receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplatto; irsogladine; isobengazole; iso-homo-halicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarazole; linear polyamine analog; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; HMG-CoA reductase inhibitor (such as, but not limited to, lovastatin, pravastatin, etc.); Petition 870260057806, dated 06 / 15 / 2026, p. 124 / 374 114 / 256 Fluvastatin, Statin, Simvastatin and Atorvastatin); loxoribine; lurtotecan; lutetium texafirin; lysophilin; lytic peptides; maytansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double-stranded RNA; mitoguazone; mitolactol; mitomycin analogs; mitonafide; mitotoxin fibroblast growth factor saponin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotropin; monophosphoryl lipid A+ cell wall of myobacteria sk; mopidamol; multidrug resistance gene inhibitor; tumor therapy based on multiple tumor suppressor 1; mustard anticancer agent; micaperoxide B; microbial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; nafterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrulin; O6-benzylguanine; octreotide; oquicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palau'amine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; Pazeliptin; pegaspargase; peldesine; pentosan sodium polysulfate; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; platinum activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;bis; Petition 870260057806, dated 06 / 15 / 2026, p. 125 / 374 115 / 256 propyl acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxyl polyoxyethylene hemoglobin conjugate; raf antagonists; raltitrexate; ramosetron; farnesyl ras protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; demethylated reteliptin; rhenium etidronate Re 186; rhizoxin; ribozymes; RII retinamide; rogletimide; roitucine; romurtide; roquinimex; rubiginone B1; ruboxil; safingol; saintopine; SarCNU; sarcofitol A; sargramostim; Sdi 1 mimetic; semustine; senescence-derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single-chain antigen-binding protein; sizofiran; sobuzoxane; sodium borocaptate;sodium phenylacetate; solverol; somatomedin-binding protein; sonermine; asparfosic acid; spikemycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell vision inhibitors; stipiamide; stromelysin inhibitors; sulfinsin; overactive vasoactive intestinal peptide antagonist; suradist; suramin; swainsonine; synthetic glycosaminoglycans; talimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogallan sodium; tegafur; telurapyrylium; telomerase inhibitors; temoporphine; temozolomide; teniposide; tetrachlordecaoxide; tetrazomine; taliblastine; thiocoralin; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid-stimulating hormone; ethyl tin ethiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; Petition 870260057806, dated 06 / 15 / 2026, p. 126 / 374 116 / 256 triacetyluridine; tricyribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tirrostines; UBC inhibitors; ubenimex; urogenital sinus-derived growth factor inhibitor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltin; Vitaxin®; vorozol; zanotron; zeniplatin; zilascorb; and zinnostatin estimalamer. Additional anticancer drugs are 5-fluorouracil and leucovorin. These two agents are particularly useful when used in methods employing thalidomide and a topoisomerase inhibitor. In some embodiments, the present disclosure DLL3-targeting trispecific protein is used in combination with gemcitabine. In some embodiments, the DLL3-targeting trispecific protein, as described herein, is administered before, during, or after surgery. Methods for detecting DLL3 expression and diagnosing DLL3-associated cancer

[0240] According to another embodiment of the disclosure, kits are provided to detect DLL3 expression in vitro or in vivo. The kits include the DLL3-binding proteins, trispecific proteins targeting DLL3 (e.g., a trispecific protein containing a labeled single-domain anti-DLL3 antibody or antigen-binding fragments thereof) previously mentioned, and one or more compounds to detect the marker. In some embodiments, the labeling is selected from the group consisting of a fluorescent label, an enzymatic label, a radioactive label, an active nuclear magnetic resonance label, a luminescent label, and a chromophore label.

[0241] In some cases, DLL3 expression is detected in a biological sample. The sample can be any sample, that Petition 870260057806, dated 06 / 15 / 2026, p. 127 / 374 117 / 256 includes, but is not limited to, tissue from biopsies, autopsies, and pathology specimens. Biological samples also include tissue sections, for example, frozen sections taken for histological purposes. Biological samples additionally include body fluids such as blood, serum, plasma, sputum, spinal fluid, or urine. A biological sample is typically obtained from a mammal, such as a human or non-human primate.

[0242] In one embodiment, a method is provided for determining whether an individual has cancer by placing a sample from the individual in contact with a single-domain anti-DLL3 antibody or a trispecific anti-DLL3 protein, as disclosed herein; and detecting the binding of the single-domain antibody to the sample. An increase in antibody binding to the sample compared to antibody binding to a control sample identifies the individual as having cancer.

[0243] In another embodiment, a method is provided for confirming a cancer diagnosis in an individual by placing a sample from an individual diagnosed with cancer in contact with a single-domain anti-DLL3 antibody or a trispecific anti-DLL3 protein as disclosed in this document; and detecting the antibody binding to the sample. An increase in antibody binding to the sample compared to antibody binding to a control sample confirms the cancer diagnosis in the individual.

[0244] 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 methods additionally include placing a second antibody that specifically binds to a single-domain anti-DLL3 antibody or a trispecific anti-DLL3 protein in contact with the sample; and detecting the binding of the second antibody. An increase in binding of the second antibody to the sample in Petition 870260057806, dated 06 / 15 / 2026, p. 128 / 374 118 / 256 Comparison with the binding of the second antibody to a control sample detects cancer in the individual or confirms a cancer diagnosis in the individual. In some cases, the cancer is a neuroendocrine cancer, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, 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 an individual without cancer. In particular examples, the sample is a blood or tissue sample.

[0245] In some cases, the antibody that binds (e.g., specifically binds) to DLL3 is directly labeled with a detectable label. In another embodiment, the antibody that binds (e.g., specifically binds) to DLL3 (the first antibody) is unlabeled, and a second antibody or other molecule that can bind to the antibody that specifically binds to DLL3 is labeled. A second antibody is chosen so that it is capable of specifically binding to the specific species and class of the first antibody. For example, if the first antibody is a llama IgG, then the secondary antibody could be an anti-llama IgG. Other molecules that can bind to antibodies include, without limitation, protein A and protein G, both of which are commercially available.Suitable identifications for the antibody or secondary 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, beta-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, and isothiocyanate. Petition 870260057806, dated 06 / 15 / 2026, page 129 / 374 119 / 256 fluorescein, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting example of a luminescent material is luminol; a non-limiting example of a magnetic agent is gadolinium, and non-limiting examples of radioactive tracers include 125I, 131I, 35S, or 3H.

[0246] In an alternative embodiment, DLL3 can be assayed in a biological sample by a confirmatory immunoassay that uses DLL3 standards labeled with a detectable substance and an unlabeled antibody that specifically binds to DLL3. In this assay, the biological sample, the labeled DLL3 standards, and the antibody that specifically binds to DLL3 are combined, and the amount of labeled DLL3 standard bound to the unlabeled antibody is determined. The amount of DLL3 in the biological sample is inversely proportional to the amount of labeled DLL3 standard bound to the antibody that specifically binds to DLL3.

[0247] The immunoassays and method disclosed herein can be used for numerous purposes. In one embodiment, the antibody that specifically binds to DLL3 can be used to detect DLL3 production 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 DLL3 soluble in a body fluid sample, such as a blood or serum sample).

[0248] In one embodiment, a kit is provided to detect DLL3 in a biological sample, such as a blood sample or tissue sample. For example, to confirm a cancer diagnosis in an individual, a biopsy may be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample may be obtained to Petition 870260057806, dated 06 / 15 / 2026, page 130 / 374 120 / 256 detect the presence of soluble DLL3 protein or fragment. Kits for detecting a polypeptide will typically comprise a single-domain antibody, according to the present disclosure, that specifically binds to DLL3. In some embodiments, an antibody fragment, such as an scFv fragment, a VH domain, or a Fab, is included in the kit. In a further embodiment, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzymatic label).

[0249] In one embodiment, a kit includes instructional materials that disclose means of using an antibody that binds to DLL3. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disc) or may be visual (such as video files), or provided via an electronic network, for example, the internet, the World Wide Web, an intranet, or another network. Kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic identifications, filter sets for detecting fluorescent identifications, appropriate secondary identifications such as a secondary antibody, or the like). Kits may additionally include buffers and other reagents routinely used for practicing a particular method.These kits and appropriate contents are well known to those skilled in the art.

[0250] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassays may vary with the particular format employed, the method for detecting DLL3 in a biological sample generally includes the steps of contacting the biological sample with an antibody that reacts specifically, under immunologically reactive conditions, to a DLL3 polypeptide. The antibody is allowed to bind specifically under immunologically reactive conditions to Petition 870260057806, dated 06 / 15 / 2026, page 131 / 374 121 / 256 form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0251] Methods for determining the presence or absence of a cell surface marker are well known in the art. For example, antibodies can be conjugated to other compounds including, but not limited to, enzymes, magnetic microspheres, colloidal magnetic microspheres, haptens, fluorochromes, metallic compounds, radioactive compounds, or drugs. Antibodies can also be used in immunoassays such as, but not limited to, radioimmunoassays (RIAs), ELISA, or immunohistochemical assays. Antibodies can also be used for fluorescence-activated cell sorting (FACS). FACS employs a plurality of color channels, obtuse and small-angle light diffusion detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells (see U.S. Patent No. 5,061,620).Any of the single-domain antibodies that bind to DLL3, as disclosed herein, can be used in these assays. Thus, the antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, a RIA, FACS, tissue immunohistochemistry, Western blot, or immunoprecipitation. EXAMPLES. Example 1: Phage Display Library Screening for Identifying DLL3 Binding Domains

[0252] Llamas were immunized with purified DLL3 protein expressed in EXPI293™ cells. A phage display library for expression of variable heavy chain antibody domains was constructed from circulating B cells (see van der Linden, de Geus, Stok, Bos, van Wassenaar, Verrips, and Frenken. 2000. J Immunol Methods 240:185–195). Phage clones were screened for DLL3 binding by expressing the clones in E. coli, preparing extracts Petition 870260057806, dated 06 / 15 / 2026, page 132 / 374 122 / 256 periplasmic clones were screened for DLL3 binding activity by ELISA. Fifty-two single-domain, single-heavy-chain antibodies were identified as producing a signal in the ELISA screening (SEQ ID Nos. 1 to 52). The CDR1, CDR2, and CDR3 sequences for these variable heavy domains were, respectively, SEQ ID Nos. 443 to 494, SEQ ID Nos. 885 to 936, and SEQ ID Nos. 1327 to 1378. Example 2: Humanization of DLL3-Binding Single Domain Antibodies and T-Cell Dependent Cellular Cytotoxicity Assay

[0253] Thirty-four (SEQ ID Nos. 53 to 86) exemplary single-domain heavy chain anti-Llama DLL3 antibodies from Example 1 were humanized. The CDR1, CDR2, and CDR3 sequences for the 34 single-domain heavy chain antibodies were, respectively, SEQ ID Nos. 495 to 528, SEQ ID Nos. 937 to 970, and SEQ ID Nos. 1379 to 1412.

[0254] The humanized anti-DLL3 sequences were cloned into an expression vector, in an expression construct comprising a signal domain followed by a single variable anti-DLL3 heavy chain domain followed by a GGGGSGGGS ligand (SEQ ID No. 1808) followed by a single-domain anti-human albumin 10G antibody (SEQ ID No. 1774) followed by a GGGGSGGGS ligand (SEQ ID No. 1808) followed by an anti-human CD3 2B2 antibody (SEQ ID No. 1793) followed by an HHHHHH tag (SEQ ID No. 1819), to generate trispecific anti-DLL3 constructs.

[0255] The anti-DLL3 trispecific constructs containing the humanized anti-DLL3 binding sequences were then transfected into EXPI293™ cells. These anti-DLL3 trispecific constructs have a genetic modification with a protein A binding site, and the amount of anti-DLL3 trispecific construct in the conditioned media of the transfected EXPI293™ cells was quantified using a Petition 870260057806, dated 06 / 15 / 2026, page 133 / 374 123 / 256 Octet instrument with protein A tips. A trispecific protein of similar molecular weight as the anti-DLL3 trispecific proteins was used as a standard.

[0256] Using conditioned media containing known concentrations of trispecific anti-DLL3 proteins, the binding affinities of trispecific anti-DLL3 proteins to human and cynomolgus monkey DLL3 proteins were measured using a method in which DLL3 proteins were expressed as human IgG1-Fc fusions and measurements were performed using an Octet instrument with anti-human Fc tips. KD measurements were obtained using a single 50 nM concentration of trispecific anti-DLL3 proteins, allowing for ranking based on potency. The relative affinities, measured as described above, are listed in Table 1. All sequences were found to bind to human DLL3, with relative affinities (KD) ranging from 0.5 to 42 nM.It was found that some of the sequences bind to cynomolgus DLL3 with affinities similar to human DLL3, and the relative affinities for the binding of such sequences to cynomolgus DLL3 are also shown in Table 1.

[0257] The conditioned media were also tested in a T cell-dependent cellular 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 carcinoma cell line; ATCC No. ATCC® CRL-2064™) were matched with purified human T cells from a donor, and a titer of the anti-DLL3 trispecific proteins was tested.

[0258] It was assumed that if a trispecific antiDLL3 protein directed T cells to kill DLL3-expressing DMS-153 cells, then the viability of the DMS-153 cells, as determined by running a luciferase assay at 48 hours Petition 870260057806, dated 06 / 15 / 2026, page 134 / 374 124 / 256 after the start of the experiment, it should decrease.

[0259] As illustrated in Figs. 2-6, which show representative TDCC data plots, several exemplary trispecific anti-DLL3 proteins were able to decrease the viability of DMS-153 cells. Fig. 2 shows TDCC assay results for trispecific anti-DLL3 proteins comprising DLL3-binding domains DH18 (SEQ ID No. 59), DH11 (SEQ ID No. 55), DH67 (SEQ ID No. 42), and DH56 (SEQ ID No. 73). Fig. 3 shows TDCC assay results for trispecific anti-DLL3 proteins comprising DLL3-binding domains DH2 (SEQ ID No. 60), DH43 (SEQ ID No. 68), DH10 (SEQ ID No. 54), and DH6 (SEQ ID No. 75). Figure 4 shows results from the TDCC assay for the trispecific protein DLL3, which comprises DLL3-binding domains DH82 (SEQ ID No. 81), DH23 (SEQ ID No. 62), DH89 (SEQ ID No. 84), and DH17 (SEQ ID No. 58).Figure 5 shows results from the TDCC assay for the trispecific protein DLL3 comprising DLL3-binding domains DH83 (SEQ ID No. 82), DH12 (SEQ ID No. 56), DH61 (SEQ ID No. 76), and DH29 (SEQ ID No. 64). Figure 6 shows results from the TDCC assay for the trispecific protein DLL3 comprising DLL3-binding domains DH58 (SEQ ID No. 74) and DH70 (SEQ ID No. 79). A negative control for the TDCC assays was a trispecific protein targeting GFP instead of DLL3 (as shown in Figure 6) which did not direct T cells to kill DMS-153 cells. EC50 values ​​from the TDCC assay are also listed in Table 1. These values ​​are in a range of 69 pM to 11 nM. Table 1: Activity of Humanized Anti-DLL3 Trispecific Proteins in DMS-153 TDCC Assays and Their Affinities with Human and Cynomolgus DLL3 Protein. Kd measurements were obtained using a single concentration of anti-DLL3 trispecific protein. The TDCC assay was performed using human T cells. n / d indicates that binding was not detected. Petition 870260057806, dated 06 / 15 / 2026, page 135 / 374 125 / 256 Ligante DLL3 DMS-153 TDCC EC50 (M) huDLL3 KD (nM) cyDLL3 KD (nM) DH43 6.9E-11 4.3 5.5 DH12 7.8E-11 1.3 n / d DH11 9.3E-11 5.3 5.6 DH58 1.1E-10 3.3 27.9 DH6 1.2E-10 6.1 6.8 DH83 1.5E-10 4.7 n / d DH10 1.6E-10 3.9 25.0 DH17 1.6E-10 7.0 n / d DH67 2.0E-10 8.4 8.2 DH2 2.6E-10 6. 5 14, 6 DH56 3,4E-10 8,1 8, 0 DH70 3,4E-10 16, 2 86, 2 DH61 3,8E-10 10, 6 30, 8 DH89 4,0E-10 6, 9 n / d DH23 4,0E-10 9, 9 n / d DH2 9 4,2E-10 5, 6 n / d DH5 5,2E-10 0, 5 5, 5 DH18 6,4E-10 1,0 5, 9 DH45 6,9E-10 1, 9 2,8 DH82 8,4E-10 6, 6 n / d DH80 1,0E-09 0, 8 5, 5 DH27 1,2E-09 2,1 11, 3 DH69 1,4E-09 1,2 7,0 DH92 1,7E-09 18, 0 17,5 DH94 1,8E-09 2, 6 9, 6 DH42 1,8E-09 4,3 11, 7 DH1 2,0E-09 3, 5 10, 7 DH38 2,9E-09 11, 9 n / d DH51 3,8E-09 5,1 18, 2 DH54 4,5E-09 20, 6 42,4 Petition: 870260057806, on June 15, 2026, page. 136 / 374 126 / 256 Ligante DLL3 DMS-153 TDCC EC50 (M) huDLL3 KD (nM) cyDLL3 KD (nM) DH3 6, 2E-09 41, 9 n / d DH15 2,0E-08 17,4 n / d DH22 2,8E-08 6, 8 16, 4 DH84 1,1E-08 15, 2 17, 9 Example 3: Phage Display Library Screening for Identifying DLL3 Linking Domains with Affinities of Superior Linkage, Using Two Domain Antibodies Simple humanized DLL3 from the previous example

[0260] Two of the humanized antibody sequences, DH43 (SEQ ID No. 68) and DH6 (SEQ ID No. 75) were used as starting points to produce phage display libraries (following a method as described in document no. WO2016187101A2). The anti-DLL3 sequences from this positioning were subsequently cloned into an expression vector, in an expression construct comprising a signal domain followed by a single variable anti-DLL3 heavy chain domain followed by a GGGGSGGGS ligand (SEQ ID No. 1808) followed by a single-domain anti-human albumin antibody domain followed by a GGGGSGGGS ligand (SEQ ID No. 1808) followed by an anti-human CD3 antibody fragment followed by an HHHHHH tag (SEQ ID No. 1819), to generate trispecific anti-DLL3 proteins. These constructs were transfected into EXPI293™ cells, and the trispecific anti-DLL3 proteins expressed were quantified as described in Example 2. The sequences of the clones identified from the positioning are SEQ ID Nos.87 to 367. Table 2 provides CDR variations obtained in the DLL3 ligand DH43 sequences after phage display selection. Three of the clones identified from positioning, SEQ ID Nos. 199 (2E05), 330 (4D09), and 365 (4H011) were genetically modified to generate variants, where each variant had a single amino acid change from the . Petition 870260057806, dated 06 / 15 / 2026, page 137 / 374 127 / 256 parental sequence, for example, to remove potential metabolic risks from the parental sequence. In particular, the DLL3-binding domains comprising SEQ ID Nos. 227 (2E05M106Y), 228 (2E05-M106Q) were genetically modified variants of SEQ ID No. 199 (2E05); SEQ ID No. 366 (4D09-M34L) was a genetically modified variant of SEQ ID No. 330 (4D09); and SEQ ID No. 367 (4H11-M34L) was a genetically modified variant of SEQ ID No. 365 (4H011). The CDR1 sequences of these DLL3-binding clones identified by positioning are SEQ ID Nos. 529 to 809, the CDR2 sequences of the clones identified by positioning are SEQ ID Nos. 971 to 1251, and the CDR3 sequences of the clones identified by positioning are SEQ ID Nos. 1413 to 1691. Table 2: Variants in CDR sequences by amino acid position of DH43 and its derivatives CDR Amino Acid Position CDR Amino Acid Variants CDR1 26 G 27 A, E, F, G, I, K, L, N, Q, R, S, T, V, Y 28 A, G, I, K, P, R, S, T, V 29 A, D, F, K, L, N, P, Q, R, S, T, Y 30 A, D, F, H, I, K, L, M, N, P, R, S, T, V, Y 31 F, I, K, L, M, N, R, S, T, V 32 N 33 A, G 34 F, I, L, M, T, V, Y 35 A, G 36 W CDR2 50 G 51 I, V 52 S Petition 870260057806, dated 06 / 15 / 2026, pp. 138 / 374 128 / 256 CDR Posição de aminoácido Variantes de Aminoácido de CDR 53 A, K, P, R, S 54 D, N 55 D, E, G, K, N, Q, R, S, T, Y 56 S, T 57 A, E, F, H, I, K, L, N, Q, R, S, T, V, Y 58 A, I, L, M, V, Y 59 D, F, H, I, L, N, S, T, V, Y 60 A, D, E, F, G, I, K, L, N, Q, R, S, T, V, Y 61 A, D, E, G, K, Q, S, V 62 S 63 A, V 64 K 65 G, V CDR3 98 F, Y 99 G, H, I, K, N, R, S, T 100 A, F, H, I, K, L, M, N, P, Q, R, S, T, Y 101 A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, Y 102 A, C, D, E, G, H, I, K, L, N, P, Q, R, S, T, W, Y 103 G, K, L, R, T 104 A, G, H, L, Q, R, S, T, V, Y 105 A, D, E, G, H, P, Q, S, T, W, Y 106 A, G, I, K, L, M, N, Q, R, S, T, V, Y 107 A, G, K, P, R, S, T, V 108 A, F, S, Y

[0261] Com o uso do meio condicionado com concentrações conhecidas das proteínas triespecíficas anti-DLL3, as afinidades Petição 870260057806, de 15 / 06 / 2026, pág. 139 / 374 The binding affinity of 129 / 256 anti-DLL3 trispecific proteins to human DLL3 protein was measured using a method in which the biotinylated version of human DLL3 protein was expressed as a human IgG1 fusion protein, and the binding affinity measurement was performed on an Octet instrument with streptavidin tips. Kd measurements were obtained using a single 50 nM concentration of the anti-DLL3 trispecific proteins, allowing for potency ranking. In this experiment, the relative Kd values ​​of the affinity-matured clones ranged from 2.3 nM to 64 nM, as listed in Table 3. The parental ligands DH43 and DH6, respectively, had Kd values ​​of 7.7 ± 0.6 nM and 9.9 ± 0.3 nM based on four conditioned medium samples from four transfections.

[0262] To select DLL3 ligand molecules identified in this positioning cycle, as well as for the DH43 and DH6 ligands of parental DLL3, more precise affinity measurements for human DLL3 were produced using 60 nM, 20 nM, 6.67 nM, and 2.22 nM concentrations of the trispecific anti-DLL3 proteins. In addition, relative affinity measurements were produced using only 60 nM of the trispecific anti-DLL3 proteins. The binding affinities determined from more precise measurements of specific anti-DLL3 binding molecules are listed in Table 4 [1H012 (SEQ ID No. 162); 1A011 (SEQ ID No. 95); 2E05 (SEQ ID No. 199); 4H011 (SEQ ID No. 365); 3C04 (SEQ ID No. 251); 2E02 (SEQ ID No. 198); 2H02 (SEQ ID No. 221); 3A011(SEQ ID No. 238); 3A02 (SEQ ID No. 230); 4D09 (SEQ ID No. 330); DH43 (SEQ ID No. 68); and DH6(SEQ ID No. 75)].In this study, the parent ligand, DH43, had a KD value of 8.9 nM, while the daughter molecule with the highest affinity, 1H012 (SEQ ID No. 162), had an affinity of 2.9 nM. Additionally, 1H012 (SEQ ID No. 162) also retained an ability to bind to DLL3 cynomolgus. Also in this study... Petition 870260057806, dated 06 / 15 / 2026, p. 140 / 374 130 / 256 The parent ligand, DH6, had a Kd value of 9.0 nM, while the daughter molecule with the highest affinity, 4H011 (SEQ ID No. 365), had an affinity of 3.9 nM. Additionally, 4H011 (SEQ ID No. 365) also retained an ability to bind to DLL3 cynomolgox.

[0263] Twenty-two DLL3 ligand molecules identified in this positioning cycle were selected for testing in a TDCC assay with DMS-153 cells, using the same protocol as described in Example 2. Exemplary TDCC data are plotted as graphs in Figs. 7-11, and a summary of EC50 values ​​is listed in Table 5. In this experiment, the parental DLL3 molecules, DH43 and DH6, had EC50 values ​​of 200 nM and 340 nM, respectively. The most potent daughter molecule of DH43 was 1H012 (SEQ ID No. 162), with an EC50 value of 28 nM, demonstrating a greater than 7-fold increase in TDCC potency compared to the parental DLL3 ligand DH43. The most potent daughter molecule of DH6 was 4H011 (SEQ ID No. 365) with an EC50 value of 36 nM, therefore showing an increase of more than 8 times in TDCC potency compared to the parental DLL3 ligand molecule.Uma protein triespecifica de control que alveja GFP, usada como um controle, não teve ativadida nesse ensaio (como shown na Fig. 11). Table 3: Relative Affinities of Anti-DLL3 Trispecific Proteins Nome Kd (M) 4A010 2.3E-09 2E011 2.4E-09 1C010 2.5E-09 3H011 2.7E-09 1E011 2.7E-09 1H012 3.5E-09 4G01 3.6E-09 1A011 3.7E-09 4D01 3.7E-09 4E02 3.8E-09 2E05 3.9E-09 Nome Kd (M) 4B011 3.9E-09 1F02 4.0E-09 1A05 4.0E-09 2A011 4.0E-09 2E010 4.0E-09 2C02 4.1E-09 2E01 4.1E-09 2G08 4.1E-09 1C01 4.3E-09 4B07 4.3E-09 1E09 4.4E-09 Nome Kd (M) 2H02 4.4E-09 3F010 4.4E-09 1D011 4.4E-09 3C04 4.5E-09 4H011 4.5E-09 4D09 4.7E-09 1A012 4.9E-09 2D012 4.9E-09 3C03 4.9E-09 1F011 5.0E-09 2H011 5.0E-09 Nome Kd (M) 1D010 5.0E-09 4C01 5.1E-09 1B01 5.2E-09 1D09 5.2E-09 1E012 5.3E-09 3D011 5.3E-09 1C05 5.3E-09 2H03 5.3E-09 1B09 5.4E-09 4B09 5.4E-09 2D011 5.4E-09 Petition 870260057806, de 15 / 06 / 2026, pág. 141 / 374 131 / 256 Nome Kd (M) 2A04 5.6E-09 1A0 6 5.6E-09 4A011 5.6E-09 2G03 5.6E-09 2B07 5.7E-09 1B011 5.7E-09 1H01 5.7E-09 1E010 5.7E-09 4F010 5.8E-09 1D01 5.8E-09 1F05 5.8E-09 1D03 5.8E-09 4D011 5.8E-09 1F012 5.8E-09 3C08 5.9E-09 2F03 5.9E-09 4D08 5.9E-09 3D07 5.9E-09 2D07 6.0E-09 2E02 6.0E-09 4C011 6.0E-09 2C08 6.1E-09 1C03 6.1E-09 2H07 6.1E-09 4H04 6.1E-09 1C02 6.2E-09 2C07 6,2E-09 1H011 6,2E-09 1H07 6,2E-09 2D04 6,2E-09 3A0 9 6,3E-09 2H04 6,3E-09 1F010 6,3E-09 1A03 6,3E-09 2C09 6.4E-09 2H010 6,4E-09 4D05 6,5E-09 2G07 6,5E-09 1A010 6,5E-09 2F09 6,5E-09 2B02 6,6E-09 4C03 6,6E-09 Nome Kd (M) 1A0 9 6.6E-09 2D06 6.6E-09 1G01 6.6E-09 2C06 6.7E-09 4C02 6.8E-09 2C04 6.8E-09 3A011 6.8E-09 1G011 6.8E-09 4C06 6.8E-09 2D03 6.8E-09 1B010 6.8E-09 1D06 6.8E-09 3G010 6.9E-09 4C010 7.0E-09 1E02 7.0E-09 1A01 7.0E-09 4B02 7.1E-09 1C07 7.1E-09 3F011 7.1E-09 1E07 7.1E-09 4E08 7.2E-09 3B05 7.2E-09 2B012 7.3E-09 3G09 7.3E-09 3B07 7.3E-09 2D010 7.3E-09 2B05 7.4E-09 4D06 7.5E-09 4G011 7.5E-09 4C07 7.5E-09 3F05 7.5E-09 2C010 7.6E-09 2B03 7.6E-09 4G08 7.6E-09 1C011 7.6E-09 2A08 7.7E-09 1A04 7.8E-09 3C09 7.8E-09 2H06 7.9E-09 2G09 8.0E-09 2F07 8.0E-09 1B05 8.0E-09 Nome Kd (M) 2A01 8,0E-09 3H06 8, 0E-09 1E04 8,1E-09 1C04 8, 1E-09 3A02 8, 1E-09 2A03 8,2E-09 3G01 8,2E-09 4F011 8,2E-09 2D09 8,2E-09 3C05 8,2E-09 4C05 8,3E-09 1C06 8,3E-09 2D05 8,3E-09 1G07 8,3E-09 1H010 8,4E-09 2E09 8,5E-09 1C012 8.5E-09 1A0 7 8.6E-09 3H010 8.6E-09 4D04 8.6E-09 1B03 8.7E-09 4F09 8.8E-09 4G09 8.8E-09 3G04 8.8E-09 2A05 8.9E-09 2A0 6 8.9E-09 1F06 8.9E-09 1B07 8.9E-09 4H08 8.9E-09 4A02 9.0E-09 4F08 9. 0E-09 4E010 9.0E-09 3H01 9. 0E-09 3B011 9. 0E-09 4A0 9 9. 0E-09 4E09 9. 1E-09 3C02 9.1E-09 2F01 9.2E-09 3A0 4 9.2E-09 1D012 9.3E-09 1E08 9.4E-09 4A05 9.4E-09 Nome Kd (M) 1F01 9,4E-09 2F02 9,6E-09 1D04 9,7E-09 4G05 9,7E-09 4F04 9, 8E-09 4A0 7 9, 8E-09 4G010 9, 9E-09 4D010 9,9E-09 3H03 9,9E-09 3F06 9,9E-09 1D08 1,0E-08 2B010 1,0E-08 3B01 1,0E-08 3D01 1,0E-08 4A01 1,0E-08 2B01 1,0E-08 3C06 1,0E-08 1H02 1,0E-08 1G09 1,0E-08 4E06 1,0E-08 2F06 1,0E-08 2A0 9 1,0E-08 3E09 1,0E-08 1F04 1,0E-08 4B08 1,0E-08 2G04 1,1E-08 4B01 1.1E-08 1B02 1.1E-08 1B04 1.1E-08 2E06 1.1E-08 3E011 1.1E-08 4E01 1.1E-08 3D03 1.1E-08 4E07 1.1E-08 1G04 1.1E-08 3E04 1,1E-08 2B011 1,1E-08 3E02 1,2E-08 3D02 1,2E-08 3A010 1,2E-08 2C01 1,2E-08 3G06 1,2E-08 Petition: 870260057806, on June 15, 2026, page. 142 / 374 132 / 256 Nome Kd (M) 3B010 1,2E-08 3A03 1,2E-08 3F09 1,2E-08 4B04 1,2E-08 3G08 1,2E-08 3A08 1,2E-08 3B02 1,2E-08 4F03 1,2E-08 1B08 1,2E-08 2G011 1,3E-08 3G07 1,3E-08 4E011 1,3E-08 3H07 1,3E-08 1F07 1,3E-08 4H03 1,3E-08 4A0 6 1,3E-08 Nome Kd (M) 3F03 1.3E-08 3C011 1.4E-08 1D02 1.4E-08 1H06 1.4E-08 2D02 1.4E-08 1E05 1.4E-08 1G05 1.4E-08 3D010 1.4E-08 3F08 1.4E-08 3H09 1.4E-08 3C01 1.4E-08 3A05 1.5E-08 4F02 1.5E-08 4G02 1.5E-08 3B06 1.5E-08 4C08 1.6E-08 Nome Kd (M) 3A0 6 1.6E-08 3D05 1.6E-08 4H09 1.6E-08 4H07 1.6E-08 3A01 1.6E-08 3E01 1.6E-08 4B06 1.6E-08 1H08 1.7E-08 3G011 1.7E-08 3D08 1.7E-08 2E08 1.7E-08 4H06 1.8E-08 2H08 1.8E-08 4B05 1.8E-08 4G07 1.8E-08 3G02 2.0E-08 Nome Kd (M) 3E03 2.0E-08 2F08 2.0E-08 4G03 2.0E-08 3B09 2.0E-08 4H01 2.1E-08 3B04 2.4E-08 4A0 8 2.4E-08 1C08 2.5E-08 4D03 2.6E-08 1G06 2.6E-08 4D02 3.0E-08 1F08 3.1E-08 3D09 3.2E-08 4A0 4 3.5E-08 1F09 3.5E-08 4H05 6.4E-08 Table 4: Binding constants for human DLL3 determined using three different concentrations of trispecific anti-DLL3 proteins and binding constants for cynomolgo DLL3 determined using a simple concentration of trispecific anti-DLL3 proteins Name Human Kd (nM) Cynomolgus Kd (nM) 1H012 2.9 4.3 1A011 3.5 3.6 2E05 3.5 4.8 4H011 3.9 5.7 3C04 4.0 5.7 2E02 4.4 3.4 2H02 4.4 5.2 3A011 7.3 8.8 3A02 7.8 9.5 4D09 8.1 8.2 DH43 8.9 8.5 DH6 9.0 10 Petition 870260057806, dated 06 / 15 / 2026, page 143 / 374 133 / 256 Table 5: TDCC values ​​of DMS-153 of affinity-matured anti-DLL3 trispecific protein in conditioned medium tested in triplicate using human T cells. Name EC50 (M) 1H012 2.8E-11 2H02 3.1E-11 2E010 3.1E-11 2E05 3.3E-11 2E01 3.3E-11 4H011 3.6E-11 4E02 4.1E-11 4B011 4.8E-11 2F11 4.9E-11 4H04 5.1E-11 1A011 5.1E-11 4D09 5.2E-11 3C04 5.2E-11 2E02 5.9E-11 3D07 6.1E-11 4B07 6.7E-11 4C06 6.8E-11 2A04 8.1E-11 1C03 9.6E-11 3H06 1.2E-10 3H011 1.2E-10 2E011 1.9E-10 DH43 2.0E-10 DH6 3.4E-10 Example 4: Cloning of Bonding Molecules to DLL3 Selection of Example 3 in Mammalian Cells

[0264] Trispecific anti-DLL3 proteins described in Example 3, as well as the parental DLL3 ligand molecules, were subcloned into a CHO cell expression vector and were Petition 870260057806, dated 06 / 15 / 2026, page 144 / 374 134 / 256 stably transfected into CHO cells (see Running Deer and Allison 2004. Biotechnol. Prog. 20: 880-889). The DLL3 ligand molecules were: 2E05-M106Q (SEQ ID No. 228); 2C04 (SEQ ID No. 181); 4D09-M34L (SEQ ID No. 366); 4D09 (SEQ ID No. 330); 2E05-M106Y (SEQ ID No. 227); 1H012 (SEQ ID No. 162) (also referred to herein as 1H12); 2E05 (SEQ ID No. 199); 2H02 (SEQ ID No. 221); 4D011 (SEQ ID No. 332) (also referred to herein as 4D11); 2E02 (SEQ ID No. 198); 4H11-M34L (SEQ ID No. 367); 1A011 (SEQ ID No. 95) (also referred to herein as 1A11); DH6 (SEQ ID No. 75); and DH43 (SEQ ID No. 68). The trispecific anti-DLL3 proteins were purified after expression in CHO cells, in conditioned medium of stable clone clusters, using protein A and ion-exchange chromatography. The purified proteins were tested in a TDCC assay using the same method, as described in Example 2.The EC50 values ​​from the TDCC assay of the present example are listed in Table 6, and the data plots are shown in Figs. 12-15. The most potent molecule, 2E05-M106Q (SEQ ID No. 228), had an EC50 value of 41 nM, which is 6.6 times more potent than the parent molecule, DH43. The most potent molecule derived from DH6 was 4D09-M34L (SEQ ID No. 366), which had an EC50 value of 54 nM and is 4.4 times more potent than the parent molecule, DH6. Table 6: TDCC Activity of Affinity-Expressed and CHO-Purified Anti-DLL3 Trispecific Proteins Name EC50 (M) 2E05-M106Q 4, 10E-11 2C04 4, 30E-11 4D09-M34L 5, 40E-11 4D09 6, 00E-11 2E05-M106Y 6, 30E-11 1H12 6, 30E-11 Petition 870260057806, dated 06 / 15 / 2026, page 145 / 374 135 / 256 Name EC50 (M) 2E05 7.20E-11 2H02 9.60E-11 4D11 9.80E-11 2E02 1.20E-10 4H11-M34L 1.30E-10 1A11 1.70E-10 DH6 2.40E-10 DH43 2.70E-10 Example 5: Affinity Maturation to Obtain Enhanced Affinity Anti-DLL3 Ligands

[0265] To obtain more potent anti-DLL3 ligands, a second affinity maturation cycle was performed. Phage display libraries were created based on the parental sequences DH6 (SEQ ID No. 75) and DH58 (SEQ ID No. 74). The sequences for the ligands from this affinity maturation cycle are provided in SEQ ID Nos. 368 to 442. The DLL3 CDR1 ligand sequences identified in this affinity maturation cycle are SEQ ID Nos. 810 to 884, the DLL3 CDR2 ligand sequences identified in this affinity maturation cycle are SEQ ID Nos. 1252 to 1326, and the DLL3 CDR3 ligand sequences identified in this affinity maturation cycle are SEQ ID Nos. 1692 to 1768. Table 7 provides CDR variations obtained in the DLL3 ligand DH6 sequences after phage display selection.

[0266] The affinity-matured anti-DLL3 sequences identified as above were cloned into an expression vector, in an expression construct comprising a signal domain followed by an anti-DLL3 sequence followed by a GGGGSGGGS ligand (SEQ ID No. 1808) followed by a single-domain anti-human albumin 10G antibody (SEQ ID No. 1774) followed by a GGGGSGGGS ligand (SEQ ID No. 1808) followed by an anti-human CD3 2B2 antibody (SEQ ID No. 1793) followed by a tag Petition 870260057806, dated 06 / 15 / 2026, p. 146 / 374 136 / 256 HHHHHH (SEQ ID No. 1819), to generate anti-DLL3 trispecific constructs.

[0267] The anti-DLL3 trispecific constructs containing the affinity-matured anti-DLL3 binding sequences were then transfected into EXPI293™ cells. These anti-DLL3 trispecific constructs were subsequently genetically modified with a protein A binding site, and the amount of anti-DLL3 trispecific construct in the conditioned media of the transfected EXPI293™ cells was quantified using an Octet instrument with protein A tips. A control trispecific protein of similar molecular weight as the anti-DLL3 trispecific proteins was used as a standard.

[0268] Using conditioned medium with known concentrations of trispecific anti-DLL3 proteins, the relative binding affinities of trispecific anti-DLL3 proteins with human DLL3 protein were measured using a method in which the biotinylated version of human DLL3 protein was expressed as a human IgG1 fusion protein, and the binding affinity measurement was performed on an Octet instrument with streptavidin tips. Kd measurements were obtained using a single 50 nM concentration of trispecific anti-DLL3 protein, allowing for ranking by potency. The measured affinities are listed in Table 8. All tested sequences were found to bind to human DLL3, with Kd values ​​ranging from 0.3 nM to 34 nM.

[0269] The conditioned medium was also tested in a T cell-dependent cellular 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 DMS153 cells were matched with purified human T cells and a titration of anti-DLL3 trispecific proteins. It was hypothesized that if an anti-DLL3 trispecific protein directed T cells to kill DMS-153 cells Petition 870260057806, dated 06 / 15 / 2026, page 147 / 374 137 / 256 DLL3 expression, therefore, the viability of DMS-153 cells, as determined by running a luciferase assay 48 hours after the start of the experiment, should decrease. Fig. 16 illustrates a graph of representative TDCC data for trispecific anti-DLL3 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). EC50 values ​​from the TDCC assay are listed in Table 9. The values ​​range from 4.2 pM to 1.5 nM. A negative control for the TDCC assays was a trispecific protein that targets GFP (as shown in Fig. 16) that did not direct T cells to kill DMS-153 cells. Table 7: Variants in CDR sequences by amino acid position of DH6 and its derivatives CDR Amino Acid Position CDR Amino Acids CDR1 26 A, D, E, F, G, H, K, L, M, N, Q, R, S, V, W, Y 27 D, E, H, K, M, P, R, S, T, Y 28 A, D, G, H, K, N, P, Q, R, S, T, V, Y 29 K, S, V 30 A, F, G, H, K, L, M, N, Q, R, S, T, V, W, Y 31 D, F, H, I, K, L, M, N, Q, R, S, V, Y 32 L, M 33 S 34 I, L, M, S, T, V 35 A CDR2 50 G 51 I, V Petition 870260057806, dated 06 / 15 / 2026, pp. 148 / 374 138 / 256 CDR Amino Acid Position CDR Amino Acids 52 S 53 A, D, E, G, H, I, K, L, N, P, Q, R, S, T, V, Y 54 A, D, E, G, H, N, R, T 55 G 56 H, P, R, S 57 A, H, I, K, M, N, Q, R, T, V 58 A, D, G, H, I, L, M, N, S, T, V, Y 59 Y 60 A, F, I, L, M, R, S, T, V, Y 61 A, D, E, G, H, K, L, N, R, S, V 62 S 63 V 64 K 65 G CDR3 98 L, Y 99 D, E, G, H, K, N, Q, R, S, T, V, Y 100 Q, W 101 A, D, E, G, H, I, K, L, M, P, R, S, T, V 102 A, D, E, G, N, R, S, T, Y 103 A, P, R, S 104 A, D, F, G, H, L, M, N, Q, R, S, T, V, Y 105 A, G, I, K, P, Q, R, S, T 106 F, H, Y Table 8: Binding constants for human DLL3 determined using a single concentration of anti-DLL3 trispecific proteins. Name Kd (nM) 53A05 3.1E-10 53A04 4.2E-10 Petition 870260057806, dated 06 / 15 / 2026, p. 149 / 374 139 / 256 Nome Kd (nM) 53C04 5.0E-10 52D04 5.0E-10 53B05 6.0E-10 51G10 6.0E-10 52B01 6.1E-10 51H05 6.7E-10 53B06 7.1E-10 54B05 7.6E-10 52C04 8.2E-10 42C03 8.8E-10 51A01 9.2E-10 51E05 9.7E-10 53A09 9.7E-10 51H04 1.0E-09 42A06 1.0E-09 41H03 1.0E-09 51A05 1.1E-09 42E05 1.2E-09 51A02 1.2E-09 42D08 1.3E-09 51G02 1.3E-09 42B10 1.3E-09 42G07 1.3E-09 41D01 1.4E-09 51F03 1.4E-09 42D06 1.5E-09 41H04 1.5E-09 51B01 1.6E-09 42C08 1.8E-09 42A03 1.9E-09 42A11 2.0E-09 Petition 870260057806, de 15 / 06 / 2026, pág. 150 / 374 140 / 256 Nome Kd (nM) 42H08 2.1E-09 51A03 2.2E-09 42C11 2.3E-09 41C02 2.4E-09 51B11 2.4E-09 51F02 2.4E-09 42H05 2.7E-09 41D02 2.7E-09 42D05 2.7E-09 42E02 2.9E-09 42H11 3.1E-09 42A07 3.2E-09 42C10 3.2E-09 42B06 3.2E-09 42F08 3.2E-09 51D03 3.3E-09 41E02 3.4E-09 42G05 3.4E-09 51E02 3.5E-09 42C01 3.6E-09 42A08 3.6E-09 42E06 3.8E-09 42E07 3.9E-09 41G01 4.0E-09 42E01 4.0E-09 41D03 4.8E-09 41E01 5.3E-09 42D07 5.3E-09 42F01 5.5E-09 42C07 6.4E-09 51F04 6.7E-09 Petition 870260057806, of 15 / 06 / 2026, p. 151 / 374 141 / 256 Nome Kd (nM) 51E03 7, 2E-09 51C02 7, 5E-09 51D01 7, 9E-09 41B11 9, 9E-09 51B04 1, 6E-08 51F01 1, 6E-08 4108-1778 51G04 2, 1E-08 41F07 2, 5E-08 41D07 3, 4E-08 Table 9: DMS-153 TDCC Values ​​of Proteins Affinity-matured anti-DLL3 trispecifics in conditioned medium tested in triplicate using human T cells Name TDCC EC50 (M) 52D04 4.2E-12 51H05 5.3E-12 52B01 5.5E-12 54B05 6.2E-12 53C04 6.2E-12 51G10 6.6E-12 5.2E-12 6.8G10 53B-12 7.7E-12 52C04 8.2E-12 53A04 8.2E-12 51A02 9.5E-12 51A05 9.6E-12 53A09 9.7E-12 51E05 1.1E-11 51F003 1.5E-1H-14 1.2E-11 53B05 1.2E-11 Petition 870260057806, of 15 / 06 / 2026, p. 152 / 374 142 / 256 Nome TDCC EC50 (M) 53H04 1.3E-11 53A05 1.6E-11 51B01 1.8E-11 42D08 1.9E-11 51A01 1.9E-11 41E02 2.1E-11 41D01 2.3E-11 42C03 2.5E-11 42A03 2.5E-11 42F10 2.5E-11 51B11 2.7E-11 42A07 2.8E-11 42G07 2.8E-11 42A06 2.8E-11 42F08 3.1E-11 42E05 3.4E-11 42C01 3.5E-11 42D05 3.6E-11 41C02 3.6E-11 51D03 3.8E-11 42H05 3.8E-11 51E02 3.8E-11 42C10 3.9E-11 42D06 4.0E-11 42H08 4.0E-11 42A11 4.2E-11 41D02 4.4E-11 42A08 4.5E-11 42E02 4.7E-11 41D03 4.8E-11 41G01 5.0E-11 Petition 870260057806, de 15 / 06 / 2026, pág. 153 / 374 143 / 256 Nome TDCC EC50 (M) 42C11 5.3E-11 51A03 5.4E-11 42G05 5.9E-11 42B10 6.6E-11 42D07 8.5E-11 42F01 8.9E-11 42C08 9.4E-11 42E07 1.0E-10 42E01 1.0E-10 51C02 1.0E-10 42B06 1.1E-10 41E01 1.1E-10 51F04 1.2E-10 51F02 1.2E-10 42C07 1.3E-10 51D01 1.3E-10 42E06 1.8E-10 51F01 5.5E-10 51E03 1.4E-09 51B04 1.5E-09 Exemplo 6: Maturação por Affinidade para Obter Liganges Anti-DLL3 de Afinidade Aprimorada

[0270] Certain trispecific anti-DLL3 proteins containing DLL-3 binding sequences that had the most potent TDCC activity in the assay described in Example 5, and a trispecific anti-DLL3 protein containing the DH6 ligand of parental DLL3, were subcloned into a CHO cell expression vector and stably transfected into CHO cells (see Running Deer and Allison 2004. Biotechnol. Prog. 20: 880-889). The DLL3 binding sequences were: 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 Petition 870260057806, dated 06 / 15 / 2026, pp. 154 / 374 144 / 256 No. 429); 51X5 (SEQ ID No. 1886); 52B1 (SEQ ID No. 430); 52C4 (SEQ ID No. 431); and 52D4 (SEQ ID No. 432). The trispecific proteins were purified in conditioned medium from clusters of stable clones using protein A and ion-exchange chromatography. An SDS-PAGE image of the purified proteins is provided in Fig. 17.

[0271] Affinity measurements for human and cynomolgus DLL3 were produced using 60 nM, 20 nM, 6.67 nM, and 2.22 nM concentrations of trispecific proteins targeting biotinylated DLL3 immobilized on Octet streptavidin tips. The affinities determined from the measurements are listed in Table 10. In this experiment, trispecific anti-DLL3 containing DH6, the parental DLL3 ligand sequence for the affinity-matured DLL3 ligand sequences, had KD values ​​of 13.5 nM for human DLL3 and 11 nM for cynomolgus DLL3. In comparison, the ten trispecific anti-DLL3 proteins containing affinity-matured DLL3 ligand molecules tested in this experiment had Kd values ​​in the range of 0.9 to 2.2 nM for human DLL3 and 1.4 to 3.4 nM for cynomolgus DLL3. Thus, the enhancements were in an affinity range of 6.1 to 15 times for human DLL3 and 3.2 to 7.9 times for cynomolgus DLL3.

[0272] The purified proteins were tested in TDCC assays 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 EC50 values ​​from these TDCC assays are listed in Table 11, and graphs of the DMS-53 and DMS-153 TDCC data are provided in Figs. 18-19, respectively. The trispecific molecule targeting GFP showed no activity in these assays (as shown in Figs. 18-19). Compared to the parent molecule DH6, EC50 values ​​improved 2.3 to 12.1 times in DMS-153 cells, 4.5 to 31.5 times in NCI-H510A cells, and 8.1 to Petition 870260057806, dated 06 / 15 / 2026, page 155 / 374 145 / 256 26.1 times in DMS-153 cells. Table 10: Affinities of purified CHO expressed trispecific anti-DLL3 proteins matured by affinity for human DLL3 protein and cynomolgus in vitro Name huDLL3 Kd (nM) cyDLL3 Kd (nM) DH6 13.5 11.0 51A2 1.2 2.0 51A5 1.2 1.6 51F3 1.4 2.0 51G2 2.0 3.4 51G10 0.9 1.4 51H5 0.9 1.6 51X5 1.0 1.5 52B1 1.1 1.9 52C4 2.2 3.0 52D4 0.9 1.7 Table 11: TDCC activity of purified CHO expressed trispecific anti-DLL3 proteins matured by affinity with human cell lines and T cells DMS153, NCI-H510A and DMS53 Name DMS153 EC50 (pM) NCI- H510A EC50 (pM) DMS53 EC50 (pM) 51A2 16.7 9.1 9.8 51G2 37.7 3.7 15.9 51G10 11.0 2.3 9.6 51H5 6.0 2.4 5.4 51X5 9.0 2.8 8.3 52B1 9.1 1.3 6.5 52C4 17.9 2.0 15.9 52D4 7.2 2.5 4.9 Example 7: Cell-Dependent Cellular Cytotoxicity Assay T with the use of Trispecific Proteins that Target DLL3 Petition 870260057806, dated 06 / 15 / 2026, page 156 / 374 146 / 256 Examples that comprise a DLL3-binding protein of this revelation

[0273] Several exemplary trispecific DLL3 proteins containing a DLL3-binding domain of this discovery, 52D04 (SEQ ID NO. 432), were tested in a T cell-dependent cellular cytotoxicity (TDCC) assay (see Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27), the results are shown in Figs. 22-24. The trispecific proteins contained a DLL3-binding domain, an albumin-binding domain (anti-ALB), and a CD3-binding domain (anti-CD3), in an anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration, as shown in Fig. 20, or in an anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration, as shown in Fig. 21. The TDCC assay was performed in the presence or absence of 15 mg / ml of 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 with exemplary DLL3-binding trispecific proteins in the presence or absence of albumin.It was assumed that if a trispecific DLL3-binding protein directed T cells to kill DLL3-expressing NCI-H2171, DMS-79, SHP77, or WM2664 cells, then the viability of such cells, as determined by running a luciferase assay 48 hours after the start of the experiment, should decrease. Fig. 22 illustrates a representative TDCC data plot, using NCI-H2171 cells, for the trispecific DLL3-binding proteins in TAC or CAT configurations, which contain the following DLL3-binding domains. Fig. 23 illustrates a representative TDCC data plot, using DMS-79 cells, for the trispecific DLL3-binding proteins in TAC or CAT configurations, which contain the following DLL3-binding domains. Fig. 24 illustrates a... Petition 870260057806, dated 06 / 15 / 2026, page 157 / 374 Figure 147 / 256 illustrates a representative TDCC data graph, using SHP77 cells, for the trispecific DLL3-binding proteins in TAC or CAT configurations, containing the following DLL3-binding domains. Figure 25 illustrates a representative TDCC data graph, using WM2664 cells, for the trispecific DLL3-binding proteins in TAC or CAT configurations, containing the following DLL3-binding domains. EC50 values ​​from the TDCC assay are listed in Table 12. As shown in the graphs and indicated by the EC50 values, in the presence of human serum albumin (HSA), the trispecific DLL3-binding proteins with the CAT orientation (Fig. 21) were more potent in the TDCC assays than the trispecific DLL3-binding proteins with the TAC configuration. Table 12: TDCC activity of trispecific anti-DLL3 proteins exemplified with NCI-H2171, DMS-79, SHP77, and human T cell lines and T cells. Cell Line EC50 (pM) without HSA EC50 (pM) with HSA NCI- H2171 aDLL3:aALB:aCD3 3 224 aCD3:aALB:aDLL3 2 84 DMS-79 aDLL3:aALB:aCD3 1.1 115 aCD3:aALB:aDLL3 0.7 41 SHP7 7 aDLL3:aALB:aCD3 21* 3953 aCD3:aALB:aDLL3 11* 821 WM2664 aDLL3:aALB:aCD3 9* 855 aCD3:aALB:aDLL3 10* 422 * 15 mg / ml of bovine serum albumin (BSA) was included in these HSA assays; the aALB domain did not bind to BSA (data not shown). Example 8: Binding of trispecific proteins targeting DLL3 exemplifying human T cells.

[0274] In a cell binding study, human T cells were incubated in the presence or absence of a protein Petition 870260057806, dated 06 / 15 / 2026, pp. 158 / 374 148 / 256 trispecific protein targeting exemplary DLL3 (in the 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 additionally incubated with a secondary antibody (anti-trispecific antibody), which has the ability to recognize the anti-albumin domain in exemplary trispecific molecules conjugated to Fluorine Alexa 647. Anti-trispecific antibody binding was measured by flow cytometry. Robust anti-trispecific antibody binding was observed in the presence of exemplary DLL3 trispecific protein in the anti-DLL3:anti-ALB:anti-CD3 (TAC) configuration (right peaks in the plots in Fig. 26) compared to cells incubated with secondary antibody alone or cells incubated without exemplary trispecific proteins or secondary antibody (left peaks in the plots in Fig. 26).Robust anti-trispecific antibody binding was also observed in the presence of the exemplary DLL3 trispecific protein in the anti-CD3:anti-ALB:anti-DLL3 (CAT) configuration (right peaks in the plots in Fig. 27) compared to cells incubated with secondary antibody alone or cells incubated without exemplary trispecific proteins or secondary antibody (left peaks in the plots in Fig. 27). Example 9: Binding of exemplary trispecific proteins targeting DLL3 to DLL3-expressing cancer cell lines.

[0275] In another linkage study, cancer cells expressing DLL3 [NCI-H82 (lung cancer cell line), SHP77 (lung cancer cell line), DMS53 (lung carcinoma), or NCI-H2171 (lung cancer cell line)] were incubated with exemplary trispecific molecules targeting DLL3 (in the CAT or TAC configuration; SEQ ID No. 1890 and SEQ ID No. 1891) or a trispecific molecule targeting GFP. Petition 870260057806, dated 06 / 15 / 2026, page 159 / 374 149 / 256 control. After incubation, the cells were washed to remove unbound trispecific molecules and additionally incubated with a secondary antibody, which has the ability to recognize the anti-albumin domain in trispecific molecules, conjugated to Fluorine Alexa 647 or FITC. The binding of exemplary trispecific molecules targeting DLL3 or that of control trispecific molecules to the cells was measured by flow cytometry. Robust binding of the DLL3-targeting trispecific molecule (in the TAC configuration) to each cell line was observed (right peaks in the plots in Fig. 28) compared to cells incubated with a control GFP-targeting trispecific molecule (left peaks in the plots in Fig. 28). Robust binding of the DLL3-targeting trispecific molecule (in the CAT configuration) to each cell line was also observed (right peaks in the plots in Fig. 28).29) compared to cells incubated with a control trispecific molecule targeting GFP (left peaks in the plots in Fig. 29). In control experiments with cell lines lacking DLL3 expression, HCTI16 (colon cancer cell line) and NCIH292 (lung cancer cell line), similar amounts of anti-trispecific antibody were bound to cells incubated with the exemplary DLL3-targeting trispecific proteins or control GFP-targeting trispecific molecules (data not shown), indicating that the exemplary DLL3-targeting trispecific molecules did not bind to cells lacking DLL3 expression. Example 10: Ability of exemplary trispecific proteins targeting DLL3 to direct T-cell-mediated death of cancer cell lines expressing DLL3.

[0276] The aim of this study was to evaluate whether exemplary trispecific molecules targeting DLL3 had the ability to direct T cells to kill DLL3-expressing cell lines NCI-H82, SHP77, DMS53 and NCI-H2171. Petition 870260057806, dated 06 / 15 / 2026, page 160 / 374 150 / 256 The DLL3-expressing cells used in this study were genetically modified to express luciferase.

[0277] For the TDCC assay (T cell-dependent cellular cytotoxicity assay), T cells from four healthy donors (donor 2; donor 47; donor 81; donor 86) and DLL3-expressing cells were mixed, and varying amounts of exemplary trispecific DLL3-targeting proteins (in CAT or TAC configurations; SEQ ID No. 1890 and SEQ ID No. 1891) were added to the mixture. The mixture was incubated for 48 hours at 37 °C. As a control, parallel experiments were performed using a control trispecific GFP-targeting molecule. After 48 hours, the remaining viable DLL3-expressing cells were quantified using a luminescence assay. It was observed that trispecific molecules targeting DLL3 (in both TAC and CAT configurations) had the ability to effectively direct T cells from all four healthy donors to kill all four cell lines expressing DLL3 (see Figs.Figures 30, 31, 32, and 33 show results using the TAC configuration; see Figures 34, 35, 36, and 37 for results using the CAT configuration) while the control GFP TriTAC molecule lacked the ability to do so (also shown in Figures 30-37). EC50 values ​​are presented in Table 13 and Table 14. Additional TDCC assays were performed with DLL3-targeting TriTAC and DLL3-deficient cell lines, NCI-H292 and HCT116. It was observed that DLL3-targeting TriTAC lacked the ability to direct T cells to kill these two DLL3-deficient cell lines (data not shown). Table 13: EC50 values ​​for TDCC assays performed using exemplary trispecific proteins targeting DLL3 containing DLL3-binding domain of this revelation, 52D04, in an anti-DLL3:anti-ALB:anti-Petition configuration 870260057806, dated 06 / 15 / 2026, page 161 / 374 151 / 256 CD3 (TAC), tested in the presence of human serum albumin (HSA), using T cells from four different donors. EC50 (M) Cell Line Donor 02 Donor 47 Donor 81 Donor 8 6 NCI-H82 3.6E-11 3.3E-11 8.0E-11 1.4E-10 SHP77 2.7E-10 1.4E-10 3.8E-10 7.0E-10 DMS53 2.3E-10 2.8E-10 2.8E-10 7.7E-10 NCI-2171 4.0E-10 2.4E-10 7.5E-10 1.0E-09 Table 14: EC50 values ​​for TDCC assays performed using exemplary trispecific DLL3-targeting proteins containing a DLL3-binding domain of this revelation, 52D04, in an anti-CD3:anti-ALB:antiDLL3 (CAT) configuration, tested in the presence of human serum albumin (HSA), using T cells from four different donors. EC50 (M) Cell Line Donor 02 Donor 47 Donor 81 Donor 8 6 NCI-H82 2.0E-11 1.6E-11 4.5E-11 5.9E-11 SHP77 6.3E-11 3.6E-11 8.4E-11 1.9E-10 DMS53 7.0E-11 7.2E-11 8.0E-11 2.2E-10 NCI-2171 1.6E-10 7.6E-11 2.9E-10 3.2E-10 Example 11: DLL3-dependent activation of T cells by exemplary trispecific proteins targeting DLL3.

[0278] 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 exemplary trispecific DLL3-targeting proteins (in CAT or TAC configurations; SEQ ID No. 1890 and SEQ ID No. 1891) for 48 hours at 37 °C. T cells from the same donors were also incubated for 48 hours at 37 °C with a control trispecific molecule, GFP TriTAC, which targets GFP and NCI-H82 or DMS53 cells. After 48 hours, T cells were collected, and CD69 and CD25 expression on T cells was measured by flow cytometry. Increased CD69 or CD25 expression was detected in cells Petition 870260057806, dated 06 / 15 / 2026, page 162 / 374 152 / 256 T cells from all 4 healthy donors were increased in the presence of NCIH82 or SHP77 cells and trispecific DLL3-targeting molecules, but not in the presence of the negative control GFP TriTAC, as observed in Figs. 38-45. A parallel experiment was performed with HCT116 cells, which lack DLL3 expression. No increase in CD69 or CD25 expression was observed with trispecific DLL3 molecules tested using HCT116 cells (data not shown). Example 12: DLL3-dependent cytokine production by T cells induced by exemplary trispecific proteins targeting DLL3.

[0279] In this assay, T cells from a healthy donor and NCI-H82 or SHP77 cells were incubated with exemplary trispecific molecules targeting DLL3 (in the CAT or TAC configuration; SEQ ID No. 1890 and SEQ ID No. 1891) for 48 hours at 37 °C. T cells from the same donor were also incubated for 48 hours at 37 °C with a control trispecific molecule, GFP TriTAC, targeting GFP and NCI-H82 or DMS53 cells. After 48 hours, the conditioned media were collected, and the amount of various cytokines present in the conditioned media was measured using an electrochemiluminescent assay (Meso Scale Discovery). It was observed that IFNγ, IL-2, and TNFα were secreted into the medium in the presence of NCI-H82 or SHP77 cells and trispecific molecules targeting DLL3, but not in the presence of the TriTAC molecule targeting control GFP. For the trispecific molecule targeting DLL3 in TAC configuration: IFNγ production is shown in Figs.For the trispecific molecule targeting DLL3 in the CAT configuration: IFNγ production is shown in Figs. 46 and 47; IL-2 production is shown in Figs. 48 and 49; TNFα production is shown in Figs. 50 and 51. For the trispecific molecule targeting DLL3 in the CAT configuration: IFNγ production is shown in Figs. 52 and 53; IL-2 production is shown in Figs. 54 and 55; TNFα production is shown in Figs. 56 and 57. Petition 870260057806, dated 06 / 15 / 2026, pp. 163 / 374 153 / 256 Example 13: Inhibition of growth of NCI-H82 xenografts by exemplary trispecific DLL3-targeting proteins

[0280] For this study, 5 x 106 human T cells and 5 x 106 NCI-H82 small cell lung cancer cells were injected into mice on day 0. On days 1 to 10, mice were injected intraperitoneally every day (ip) with exemplary trispecific DLL3-targeting molecules (in CAT or TAC configurations; SEQ ID No. 1890 and SEQ ID No. 1891) at doses of 20, 100, or 500 pg / kg or negative control TriTAC targeting GFP at a dose of 500 pg / kg. Tumor volumes were measured after several days, starting on day 7 and ending on day 24. Significant inhibition of tumor growth was observed in mice injected with trispecific proteins targeting DLL3 at all doses compared to mice dosed with TriTAC targeting GFP at 500 pg / kg, as shown in Fig. 58. Example 14: Elimination of NCI-H82 xenografts by exemplary trispecific proteins targeting DLL3.

[0281] For this study, 5 x 10⁶ NCI-H82 small cell lung cancer cells were injected subcutaneously on day 0. Mice were randomized on day 8, and 2 x 10⁷ human T cells were injected per mouse. On days 9 to 18, mice were injected daily ip with exemplary trispecific molecules targeting DLL3 (in the CAT configuration; SEQ ID No. 1890) at doses of 1, 10, or 100 pg / kg or TriTAC targeting GFP as a negative control at a dose of 100 pg / kg. Tumor volumes were measured after several days, starting on day 8 and ending on day 29. Significant inhibition of tumor growth was observed in mice injected with trispecific molecules targeting DLL3 at doses of 10 and 100 pg / kg compared to mice dosed with TriTAC targeting GFP at 100 pg / kg, as shown in Fig. 59. Petition 870260057806, dated 06 / 15 / 2026, pp. 164 / 374 154 / 256 Example 15: Inhibition of growth of SHP77 xenografts by exemplary trispecific proteins targeting DLL3

[0282] For this study, 5 x 106 human T cells and 1 x 107 SHP77 small cell lung cancer cells were injected into mice on day 0. On days 1 to 10, mice were injected daily ip with trispecific molecules targeting DLL3 (in the CAT configuration; SEQ ID No. 1890) at doses of 1, 10, or 100 pg / kg or negative control TriTAC targeting GFP at a dose of 100 pg / kg. Tumor volumes were measured after several days, starting on day 6 and ending on day 28. Significant inhibition of tumor growth was observed in mice injected with trispecific molecules targeting DLL3 at doses of 10 and 100 pg / kg compared to mice dosed with TriTAC targeting GFP at 100 pg / kg, as shown in Fig. 60. Example 16: Pharmacokinetic profile of exemplary trispecific proteins targeting DLL3.

[0283] Trispecific proteins that target DLL3 have a half-life of ~3 to ~3.9 days in cynomolgus monkeys when dosed at 0.3 mg / kg

[0284] For this study, cynomolgus monkeys were injected intravenously with 0.3 mg / kg doses of exemplary trispecific DLL3-targeting molecules (in CAT or TAC configurations; SEQ ID No. 1890 and SEQ ID No. 1891), and serum samples were collected at various time points after injection. Two monkeys were injected for each dose. The amount of trispecific DLL3-targeting molecule in serum was measured using anti-idiotype antibodies recognizing the trispecific molecule in an electrochemiluminescent assay. Fig. 61 shows a plot for serum levels of trispecific DLL3-targeting molecule at various time points. The data were then used to calculate the pharmacokinetic properties of the trispecific DLL3-targeting molecules, as provided in Petition 870260057806, dated 06 / 15 / 2026, pp. 165 / 374 155 / 256 Table 15. A once- or twice-weekly human dosing schedule is contemplated based on pharmacokinetic data. Table 15: Pharmacokinetics of exemplary trispecific molecules targeting DLL3 ID Half-life (h) AUC 0-inf (h*nM) CL (L / h / kg) Vss (L / kg) 1 93.1 7210 0.000832 0.0869 2 72.4 6690 0.000896 0.0731 3 82.6 7900 0.00076 0.0767 4 77 7890 0.00076 0.0712

[0285] The trispecific protein targeting DLL3 has a half-life of ~2.8 to ~3.3 days in cynomolgus monkeys when dosed at 1 or 10 mg / kg:

[0286] For this study, cynomolgus monkeys were injected intravenously with either 1 mg / kg or 10 mg / kg doses of exemplary trispecific molecules targeting DLL3, and serum samples were collected at various time points after injection. Two monkeys were injected for each dose. The amount of DLL3-targeting TriTAC in serum was measured using anti-idiotype antibodies recognizing the TriTAC molecule in an electrochemiluminescent assay. Fig. 62 shows a plot for serum levels of DLL3-targeting trispecific molecule at various time points. The data were then used to calculate the pharmacokinetic properties of the TriTAC molecule, as provided in Table 16. The pharmacokinetic data suggest once- or twice-weekly dosing in humans. Table 16: Pharmacokinetics of exemplary trispecific molecules targeting DLL3 Dose (mg / kg) Half-life (h) Cmax (nM) AUC 0-inf (h*nM) CL (ml / h / kg) Vss (l / kg) 1 67.5 493 23,800 0.79 63, 8 10 78, 6 4,492 236,500 0, 80 71, 9 Petition 870260057806, dated 06 / 15 / 2026, pp. 166 / 374 156 / 256

[0287] Exemplary trispecific proteins targeting DLL3 were tolerated in cynomolgus monkeys when provided as a single dose of up to 10 mg / kg:

[0288] A transient increase in serum cytokine levels was observed, primarily at the 10 mg / kg dosage of exemplary DLL3 trispecific protein administration (in the CAT setting) (Fig. 63; IFNY-Fig. 63 top panel, IL-6 Fig. 63 second panel; IL-10 Fig. 63 third panel). Transient T-cell margination and T-cell activation were also observed (data not shown). In terminal and recovery euthanasia, no macroscopic findings related to DLL3 trispecific protein or organ weight differences were observed, and in recovery euthanasia, no microscopic findings related to DLL3 trispecific protein were observed.

[0289] To demonstrate that DLL3-targeting TriTAC retained directed killing activity after administration to a cynomolgus monkey, a serum sample forming the 10 mg / kg dose group collected 168 h post-dosage was tested in a DMS53 TDCC assay and compared to freshly thawed DLL3-targeting TriTAC. Identical DMS53 cell death was observed with both the serum sample and the freshly thawed protein (Fig. 64), indicating that DLL3-targeting TriTAC retains the ability to direct T cells to kill target cells 1 week after dosing in a cynomolgus monkey. Example 17: Xenograft Tumor Model

[0290] A trispecific protein targeting anti-DLL3 exemplary of this discovery is evaluated in a xenograft model.

[0291] Female NOD / scid immunodeficient mice are irradiated sublethally (2 Gy) and inoculated subcutaneously with 1x 106 NCI-H28 cells in their right dorsal flanks. Petition 870260057806, dated 06 / 15 / 2026, page 167 / 374 157 / 256 When tumors reach 100 to 200 mm3, animals are allocated into 3 treatment groups. Groups 2 and 3 (8 animals each) are injected intraperitoneally with 1.5 x 107 activated human T cells. Three days later, animals in Group 3 are subsequently treated with a total of 9 intravenous doses of exemplary DLL3 trispecific antigen-binding protein (such as 1, 10, 50, or 100 pg / kg) (qdx9d). Groups 1 and 2 are treated with vehicle only. Body mass and tumor volume are determined for 30 days.

[0292] Animals treated with trispecific proteins targeting DLL3, as exemplified in the previous examples, are expected to have a statistically significant delay in tumor growth compared to the respective vehicle-treated control group. Example 18: Proof-of-Concept Clinical Trial Protocol for Administration of an Exemplary DLL3 Trispecific Antigen-Binding Protein (Anti-DLL3 Trispecific Protein) to Patients with Neuroendocrine Cancer

[0293] This is a Phase I / II clinical trial to study an exemplary DLL3 trispecific antigen-binding protein as a treatment for a neuroendocrine cancer. Study Results:

[0294] Primary: Maximum tolerated dose of the exemplary trispecific protein that targets DLL3

[0295] Secondary: To determine if the in vitro response of trispecific target proteins associated with the Phase I DLL3 clinical response exemplary

[0296] of phase IA maximum tolerated dose of the test. (MTD) will be determined in section 1.1 The maximum tolerated dose (MTD) will be determined in section Phase I of the test. Petition 870260057806, dated 06 / 15 / 2026, pp. 168 / 374 158 / 256 1.2 Patients who meet eligibility criteria will enter the trial to evaluate the exemplary trispecific protein that targets DLL3. 1.3 The objective is to identify the highest dose of the exemplary trispecific anti-DLL3 protein that can be safely administered without serious and intractable side effects in participants. The dose given will depend on the number of participants who enrolled in the study prior to the trial and how well the dose was tolerated. Not all participants will receive the same dose. Phase II 2.1 A subsequent phase II session will be conducted at MTD with the objective of determining whether therapy with exemplary trispecific proteins targeting DLL3 results in at least a 20% response rate. Primary Outcome for Phase II --- To determine whether therapy with the exemplary DLL3-targeting trispecific protein results in at least 20% of patients achieving a clinical response (weak response, small response, partial response, or complete response).

[0297] Eligibility: The biopsy proved the neuroendocrine tumor, which is somatostatin receptor positive as demonstrated on somatostatin receptor PET.

[0298] All sites or sources are eligible.

[0299] Functional and non-functional tumors are permitted.

[0300] No candidates for surgical treatment.

[0301] ECOG performance status 0, 1 or 2.

[0302] Age > 18.

[0303] Ability to understand a written informed consent document, and willingness to sign it.

[0304] Although preferred embodiments of the present invention have been presented and described herein, it will be obvious to those skilled in the art that such Petition 870260057806, dated 06 / 15 / 2026, page 169 / 374 159 / 256 embodiments are provided by way of example only. Various variations, alterations and substitutions will now occur for those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. The following claims are intended to define the scope of the invention and the methods and structures within the scope of these claims and their equivalents are covered by them. SEQ. ID NO. number number 1 DL1 QVQLQESGGGLVQAGGSLRLSCAASGSIFSIASMGWYRQAPGKQREL VAVITSFSSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY CNARYFERTDWGQGTQVTVSS 2 DL74 DL31 VAAITSFSSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY CNARYFERTDWGQGTQVTVSS 4 DL3 QVQLQE S GGGLVQAGGSLRLS CAASE SIFSINVMAWHRQAP GKQRE L VARITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY CGAYQGLYAYWGQGTQVTVSS 5 DL80 QVQLQESGGGLVQAGGSLRLSCVASGSSFSITSMAWYRQAPGKQRDL VAAITSFGSTNYADSVKDRFTISRDNAKNTVYLQMNSLKPEDTAVYY CNGRVFDHVYWGQGTQVTVSS 6 DL18 QVQLQESGGGLVQAGGSLKLSCAASSSIFSISSMSWYRQAPGKQREL VAAITTFDYTNYADSVKGRFTISRDNAKNMMYLQMNSLKPEDTAVYL CNARAFGRDYWGQGTQVTVSS 7 DL94 QVQLQESGGGLVQAGGSLKLSCAASSSIFSISSMSWYRQAPGKQREL VAAITSFGSTNYADSVKGRFTISRDNAKNMMYLQMNSLKPEDTAVYR CNARTMGRDYWGQGTQVTVSS 8 DL17 QVQLQESGGGLVQPGGSLRLSCAASGSTLNIKIMAWHRQAPGKQRELVATLTSGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLQPEDTAVYY CGLWDGVGGAYWGRGTQVTVSS Petition 870260057806, dated 06 / 15 / 2026, pp. 170 / 374 160 / 256 SEQ. ID NO. nome sequência 9 DL46 QVQLQESGGGLVQPGGSLRISCAASGSTLNIKIMAWHRQAPGKQREL VATLTSGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLQPEDTAVYY CGLWDGVGGAYWGRGTQVTVSS 10 DL15 QVQLQESGGGLVQAGGSLRLSCAASGSTFNIKTMAWHRQAPGNQREL VATLTSGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY CGLWNGVGGAYWGRGTQVTVSS 11 DL26 QVQLQDGGGLVQPGGSLRLSCAASGSTFNIKLMAWHRQAPGNQRELV ATLTSGGNTNYADSVKGRFTISRDNASNIVYLQMNSLKPEDTAVYYC GLWDGVGGAYWGRGTQVTVSS 12 DL83 QVQLQESGGGLVQAGGSLRLSCAASGSTFNFKIMAWHRQAPGKQREL VASLTSEGLTNYRDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYY CGLWDGVGGAYWGRGTQVTVSS 13 DL5 QVQLQESGGGLVQPGGSLRLSCAASGFMFSSYSMSWYRQAPGKQREL VAAITTWGSTNYADSVKGRFTISRDNAKNTVWLQMNSLEPEDTAVYF CNARSWNNYWGQGTQVTVS S 14 DL22 QVQLQESGGGLVQVGGSLRLSCAASGFMFSSYSMSWYRQAPGKQREL VAAITSYGSTNYADSVKGRFTISRDNAKNTVWLQMNSLKPEDTAVYF CNARSWNNYWGQGTQVTVS S 15 DL85 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSHSMSWYRQAPGKQREL VAAITTYGSTNYIDSVKGRFTISRDNTKNTVYLQMNSLKPEDTAVYF CNARSWNNYWGQGTQVTVS S 16 DL69 QVQLQESGGGLVQAGGSLRLSCVASGSSFSHNTMGWYRQAPGKQRDLVARITTFGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY CNGESFGRIWYNWGQGTQVTVSS 17 DL27 QVQLQE S GGGLVQAGAS LRLTCTAS GGRF SYATMGWSRQAP GKQREM VARITSSGF STNYAD SVKGRF TISRDNAKNAVYLQMD SLKPEDTAVY YCNAQHFGTDSWGQGTQVTVSS 18 DL51 QVQLQESGGGLVQAGASLRLTCTASGSRFSYATMGWSRQAPGKQREL VARITSSGF STNYAD SVKGRF TISRDNAKNAVYLQMD SLKPEDTAVY YCNAQQFGTDSWGQGTQVTVSS 19 DL54 QVQLQESGGGLVQAGGSLRLSCAASGSTFTSNVMGWHRQAPGKQREL VANMHSGGSTNYADSVKGRFTISRDNAKNIVYLQMNNLKIEDTAVYY CRWYGIQRAEGYWGQGTQVTVSS Petition: 870260057806, on June 15, 2026, page. 171 / 374 161 / 256 SEQ. ID NO. nome sequência 20 DL11 QVQLQESGGGLVVAGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISVDGSTNYADSVKGRFTVSRDNAKNTVYLQMNSLQPEDTAVYY CYAYRWVGRDTYWGQGTQVTVSS 21 DL19 QVQLQESGGGLVVAGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISVDGSTNYADSVKGRFTISRDNAKNTVYLQMNSLQPEDTAVYY CYAYRWVGRDTYWGQGTQVTVSS 22 DL68 QVQLQESGGGLVVSGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISVDGSTNYADSVKGRFTISRDNAKNTVYLQMNSLQPEDTAAYY CYAYRWVGRDTYWGQGTQVTVSS 23 DL14 QVQLQESGGGLVVAGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISVDGSTNYADSVKGRFTISRDNAENTVYLQMNSLQPEDTAVYY CYAYRWEGRDTYWGQGTQVTVSS 24 DL67 QVQLQESGGGLVVAGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISVDGSTNYADSVKGRFTISRDNAENTVYLQMNSLQPEDTAVYY CYAYRWEGRNTYWGQGTQVTVSS 25 DL56 QVQLQESGGGLVQPGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISTDGSTNYVDSVKGRFTISRDNAKNTVYLQMNSLQPEDTAVYY CYAYRWVGRYTYWGQGTQVTVSS 26 DL13 QVQLQESGGGLVVAGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISTDGTTNYVDSVKDRFTISRDNAKNTVYLQMNSLQPEDTAAYY CYAYRWVGRDTYWGQGTQVTVSS 27 DL77 QVQLQESGGGLVVAGGSLRLSCAASGSSVSFLSIAWYRQAPGKKRELVAGISTDGTTNYVDSVKDRFTISRDNAKNTVYLQMNSLQPEDTAAYY CYAYRWVGRDTYWGQGTQVTVSS 28 DL79 QVQLQESGGGLVQAGGSLTLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISTDGTTNYVDSVKDRFTISRDNAKNTVYLQMNSLQPEDTAAYY CYAYRWVGRDTYWGQGTQVTVSS 29 DL20 QVQLQESGGGLVQAGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISTDGSTNYADSVKGRFTISEGNAKNTVDLQMNSLQPEDTAVYY CYAYRWVDRYTYWGQGTQVTVSS 30 DL41 QVQLQESGGGLVVAGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISTDGSTNYADSVKGRFTISEDNAKNTVDLQMNSLQPEDTAVYY CYAYRWIDRYTYWGQGTQVTVSS Petição 870260057806, de 15 / 06 / 2026, pág. 172 / 374 162 / 256 SEQ. ID NO. nome sequência 31 DL59 QVQLQESGGGLVQPGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISTDGSTNYADSVKGRFTISEDNAKNTVDLQMNSLQPEDTAVYY CYAYRWVDRYTYWGQGTQVTVSS 32 DL16 QVQLQESGGGLVVAGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISSDGSTNYVDSVKGRFTISRDNAKNIVFLQMNSLQPQDTAVYY CYAYRWVGRDTYWGQGTQVTVSS 33 DL6 QVQLQESGGGLVVAGGSLRLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISADGSTDYIDSVKGRFTISRDSANNTMYLQMNSLQPEDTAVYY CYAYRWTTRYTYWGQGTQVTVSS 34 DL84 QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWYRQAPGKKREL VAGISSDGSTHYVDSVKGRFAISRDNAENTVYLQMNDLQPDDTAVYY CYAYRWVGGYTYWGQGTQVTVSS 35 DL2 QVQLQESGGGLVQAGGSLRLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGSKNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY CYYFRTVAASSMQYWGQGTQVTVSS 36 DL43 QVQLQESGGGLVQAGGSLRLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVFY CYYFRTVSGSSMRYWGQGTQVTVSS 37 DL92 QVQLQESGGGLVQAGGSLRLSCAASGITSSVYSMGWYRQAPGKQREL VAGSSSDGSTHYVDSVRGRFTISRDNAKNTVYLQMSSLKPEDTAVYY CYANRGFAGAP SYWGQGTQVTVS S 38 DL10 QVQLQE S GGGLVQAGGSLRLSCAAS GRTSMFNSMGWHRQAP GKQRELVAIIRSGGSSNYADTVKGRFTISRDNTKNTVYLQMNDLKPEDTAVYY CFYYFQSSYWGQGTQVTVSS 39 DL82 QVQLQE S GGGLVQAGGSLRLSCAAS GRTSMVNSMGWHRQAP GKQREL VALITSGGSSNYADTVKGRFTISRDNTKNTVYLQMNDLKPEDTAVYY CFYYFQSSYWGQGTQVTVSS 40 DL23 QVQLQESGGGLVQAGGSLRLSCAASGSVSMFNSMGWHRQPPGKQREL VAIITSGGSSNYADTVKGRFTISRDNTKNTVYLQMNDLKPEDTAVYY CFYYFQSSYWGQGTQVTVSS 41 DL42 QVQLQESGGGLVQAGGSLRLSCTASGSSIFSIAVMGWYRQVPGKRREW VATIFDGSYTNYADSVKGRFTISRDNARNKVYLQMNNLKPEDTAVYY CQTHWTQGSVPKESWGQGTQVTVSS Petition 870260057806, of 15 / 06 / 2026, p. 173 / 374 163 / 256 SEQ. ID NO. nome sequência 42 DL45 QVQLQESGGGLVQAGGSLRLSCVASSGIFSDMSMVWYRQAPGKQREL VASITTFGSTNYADPVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY CSGRSYSSDYWGRGTQVTVSS 43 DL58 QVQLQE S GGGLVQAGGSLRLSCVASGSISSIIVMGWSRQAP GKQRE S VATITRDGTRNYADSLKGRFTISRDNAKNTSYLQINSLKPEDTAVYS CYARYGDINYWGKGTQVTVSS 44 DL70 QVQLQE S GGGLVQAGGSLRLSCVASGSISSIIVMGWSRQAP GKQRE S LATISRGGTRTYADSVKGRFTISRDNAKNTSYLQMNSLKPEDTAVYS CYARYGDINYWGKGTQVTVSS 45 DL89 QVQLQESGGGLVQAGGSLRLSCVASGSIFTTNSMGWHRQGPGKQREL VALIGSAGSTKYADSVKGRFTISRDNAKNTVSLQMDSLKPEDTAVYY FAYDNINAYWGQGTQVTVSS 47 DL52 QVQLQESGGGWVQAGGSLRLSCAASGSREISTMGWHRQAPGKQRELA ARITSGGITKYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTAVYYC FAYDNINAYWGQGTQVTVSS 48 DL64 QVQLQESGGGWVQAGGSLRLSCTASGSREISTMGWHRQAPGKQRELA ARITSGGITKYADSVKGRFTISRDNAKKTVYLQMDSLKSEDTAVYYC FAYDNINAYWGQGTQVTVSS 49 DL33 QVQLQESGGGSVQAGRSLGLSCAASGSREISTMGWHRQAPGKQRELAARITSGGITKYADSVKGRFTISRDNAKKTVYLQMNSLKSEDTAVYC FAYDNINAYWGQGTQVTVSS 50 DL12 QVQLQESGGGLVQAGGSLRLSCTASGSIFRGAAMYWHRQAPGKQREL VAAITTSGNTSYADSVKGRFTISRDNAKNTMYLQIISLKPEDTAVYY CAFWIAGKAYWGQGTQVTVSS 51 DL29 QVQLQESGGGLVQPGGSLRLSCAASGSSFNFMSWHRQAPGKEREL AGVITRGGATNYADSVKGRFTISVNTVLQLQLQLQLQVTV CHGRSQLGSTWGQGTQVTVSS 52 DL61 QVQLQESGGGLVQAGGSLRLSCLASGTIFTAS™GWHRQPPGKQREL VASIAGDGRTNYAESTEGRFTISRDDAKNTMYLQMNSLKPEDTAVYY CYAYYLD TYAYWGQGT SQVTVS Petition 870260057806, of 15 / 06 / 2026, p. 174 / 374 164 / 256 SEQ. ID NO. nome sequência 53 DH1 EVQLVESGGGLVQPGGSLTLSCAASGSIFSIASMGWYRQAPGKQREL VAVITSFSSTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CNARYFERTDWGQGTLVTVSS 54 DH10 EVQLVESGGGLVQPGGSLTLSCAASGRTSMFNSMGWHRQAPGKQREL VAIIRSGGSSNYADTVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CFYYFQSSYWGQGTLVTVSS 55 DH11 EVQLVESGGGLVQPGGSLTLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISVDGSTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYAYRWVGRDTYWGQGTLVTVSS 56 DH12 EVQLVESGGGLVQPGGSLTLSCTASGSIFRGAAMYWHRQAPGKQREL VAAITTSGNTSYADSVKGRFTISRDNAKNSMYLQMNSLRAEDTAVYY CAFWIAGKAYWGQGTLVTVSS 57 DH15 EVQLVESGGGLVQPGGSLTLSCAASGSTFNIKTMAWHRQAPGNQREL VATLTSGGNTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CGLWNGVGGAYWGQGTLVTVSS 58 DH17 EVQLVESGGGLVQPGGSLTLSCAASGSTLNIKIMAWHRQAPGKQREL VATLTSGGNTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CGLWDGVGGAYWGQGTLVTVSS 59 DH18 EVQLVESGGGLVQPGGSLTLSCAASSSIFSISSMSWYRQAPGKQREL VAAITTFDYTNYADSVKGRFTISRDNAKNSMYLQMNSLRAEDTAVYY CNARAFGRDYWGQGTLVTVSS 60 DH2 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQRELVAGISSDGSKNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVAASSMQYWGQGTLVTVSS 61 DH22 EVQLVESGGGLVQPGGSLTLSCAASGFMFSSYSMSWYRQAPGKQREL VAAITSYGSTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY GGRF SYATMGWSRQAP GKQREM VARITSSGF STNYAD SVKGRF TISRDNAKNSVYLQMN SLRAEDTAVY YCNAQHFGTDSWGQGTLVTVSS Petition: 870260057806, on June 15, 2026, page. 175 / 374 165 / 256 SEQ. ID NO. nome sequência 64 DH2 9 EVQLVESGGGLVQPGGSLTLSCAASGSISSFNFMSWHRQAPGKEREL AGVITRGGATNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CHGRSQLGSTWGQGTLVTVSS 65 DH3 EVQLVESGGGLVQPGGSLTLSCAASESIFSINVMAWHRQAPGKQREL VARITSGGSTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CGAYQGLYAYWGQGTLVTVSS 66 DH38 EVQLVESGGGLVQPGGSLTLSCAASGSREISTMGWHRQAPGKQRELA ARITSGGITKYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYC FAYDNINAYWGQGTLVTVSS 67 DH42 EVQLVESGGGLVQPGGSLTLSCTASGSIFSIAVMGWYRQVPGKRREW VATIFDGSYTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CQTHWTQGSVPKESWGQGTLVTVSS 68 DH43 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRYWGQGTLVTVSS 69 DH45 EVQLVESGGGLVQPGGSLTLSCVASSGIFSDMSMVWYRQAPGKQREL VASITTFGSTNYADPVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CSGRSYSSDYWGQGTLVTVSS 70 DH5 EVQLVESGGGLVQPGGSLTLSCAASGFMFSSYSMSWYRQAPGKQREL VAAITTWGSTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CNARSWNNYWGQGTLVTVS S 71 DH51 EVQLVESGGGLVQPGGSLTLSCTASGSRFSYATMGWSRQAPGKQREL VARITSSGF STNYAD SVKGRFTISRDNAKNSVYLQMN SLRAEDTAVY YCNAQQFGTDSWGQGTLVTVSS 72 DH54 EVQLVESGGGLVQPGGSLTLSCAASGSTFTSNVMGWHRQAPGKQREL VANMHSGGSTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CRWYGIQRAEGYWGQGTLVTVSS 73 DH5 6 EVQLVESGGGLVQPGGSLTLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISTDGSTNYVDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYAYRWVGRYTYWGQGTLVTVSS 74 DH58 EVQLVESGGGLVQPGGSLTLSCVASGSISSIIVMGWSRQAPGKQRES VATITRDGTRNYADSLKGRFTISRDNAKNSSYLQMNSLRAEDTAVYY CYARYGDINYWGQGTLVTVSS Petition 870260057806, dated 06 / 15 / 2026, pp. 176 / 374 166 / 256 SEQ. ID NO. nome sequência 75 DH6 EVQLVESGGGLVQPGGSLTLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISADGSTDYIDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYAYRWTTRYTYWGQGTLVTVSS 76 DH61 EVQLVESGGGLVQPGGSLTLSCLASGTIFTASTMGWHRQPPGKQREL VASIAGDGRTNYAESTEGRFTISRDNAKNSMYLQMNSLRAEDTAVYY CYAYYLDTYAYWGQGTLVTVSS 77 DH67 EVQLVESGGGLVQPGGSLTLSCAASGSSVSFLSMAWYRQAPGKKREL VAGISVDGSTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYAYRWEGRNTYWGQGTLVTVSS 78 DH69 EVQLVESGGGLVQPGGSLTLSCVASGSSFSHNTMGWYRQAPGKQRDL VARITTF GTTNYAD SVKGRF TIS RDNAKN SVYLQMNSLRAE D TAVYY CNGESFGRIWYNWGQGTLVTVSS 79 DH70 EVQLVESGGGLVQPGGSLTLSCVASGSISSIIVMGWSRQAPGKQRES LATISRGGTRTYADSVKGRFTISRDNAKNSSYLQMNSLRAEDTAVYY CYARYGDINYWGQGTLVTVSS 80 DH80 EVQLVESGGGLVQPGGSLTLSCVASGSSFSITSMAWYRQAPGKQRDL VAAITSFGSTNYADSVKDRFTISRDNAKNSVYLQMNSLRAEDTAVYY CNGRVFDHVYWGQGTLVTVSS 81 DH82 EVQLVE S GGGLVQPGGSLTLSCAAS GRTSMVNSMGWHRQAP GKQREL VALITSGGSSNYADTVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CFYYFQSSYWGQGTLVTVSS 82 DH83 EVQLVESGGGLVQPGGSLTLSCAASGSTFNFKIMAWHRQAPGKQRELVASLTSEGLTNYRDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CGLWDGVGGAYWGQGTLVTVSS 83 DH84 EVQLVESGGGLVQPGGSLTLSCAASGFTLDYYAIGWYRQAPGKKREL VAGISSDGSTHYVDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYAYRWVGGYTYWGQGTLVTVSS 84 DH89 EVQLVESGGGLVQPGGSLTLSCVASGSIFTTNSMGWHRQGPGKQREL VALIGSAGSTKYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CFYYDSRSYWGQGTLVTVSS 85 DH92 EVQLVESGGGLVQPGGSLTLSCAASGITSSVYSMGWYRQAPGKQREL VAGSSSDGSTHYVDSVRGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYANRGFAGAP SYWGQGTLVTVS S Petition 870260057806, dated 06 / 15 / 2026, page 177 / 374 167 / 256 SEQ. ID NO. nome sequência 86 DH94 EVQLVESGGGLVQPGGSLTLSCAASSSIFSISSMSWYRQAPGKQREL VAAITSFGSTNYADSVKGRFTISRDNAKNSMYLQMNSLRAEDTAVYY CNARTMGRDYWGQGTLVTVSS 87 1A01 EVQLVESGGGLVQPGGSLTLSCVASGFTSSINAMGWYRRAPGKQREL VAGISSDGSFVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRHVSGSSMRYWGQGTLVTVSS 88 1A03 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSSRYWGQGTLVTVSS 89 1A0 4 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGSKVYEDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRYWGQGTLVTVSS 90 1A05 EVQLVESGGGLVQPGGSLTLSCVASGSPSSINAMGWYRRAPGKQREL SAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVRGSSMSYWGQGTLVTVSS 91 1A0 6 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL AAGISSDGSSVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSKRYWGQGTLVTVSS 92 1A0 7 EVQLVESGGGLVQPGGSLTLSCVASGSISSINAMGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRMVSGSSMRYWGQGTLVTVSS 93 1A0 9 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQRELVAGISSDGSKLYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVQGSSMRYWGQGTLVTVSS 94 1A010 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAYGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVYGSSMRYWGQGTLVTVSS 95 1A011 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAIGWYRRAPGKQREL VAGISSDGSKVYIDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSYRYWGQGTLVTVSS 96 1A012 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYSDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVLGSSMRYWGQGTLVTVSS Petition 870260057806, dated 06 / 15 / 2026, pp. 178 / 374 168 / 256 SEQ. ID NO. nome sequência 97 1B01 EVQLVESGGGLVQPGGSLTLSCVASGSTSIINAMGWYRRAPGKQREL AAGISSDGSKVIADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRRVSGSSMRYWGQGTLVTVSS 98 1B02 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGSKIYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRYWGQGTLVTVSS 99 1B03 EVQLVESGGGLVQPGGSLTLSCVASGKTSSINAMAWYRRAPGKQREL VAGISSDGSKVYTDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSARYWGQGTLVTVSS 100 1B04 EVQLVESGGGLVQPGGSLTLSCVASGTTSSINAMGWYRRAPGKQREL VAGISSDGSLVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRIVRGSSMRYWGQGTLVTVSS 101 1B05 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYYRTVSGSSMRYWGQGTLVTVSS 102 1B07 EVQLVESGGGLVQPGGSLTLSCVASGSGSSINAMGWYRRAPGKQREL VAGISSDGSKVYSDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRHVSGSSMRYWGQGTLVTVSS 103 1B08 EVQLVESGGGLVQPGGSLTLSCVASGTTSSINAMGWYRRAPGKQREL VAGISSDGSKVYVDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRFVSGSSMRYWGQGTLVTVSS 104 1B09 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQRELVAGISSDGSKVYVDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRYWGQGTLVTVSS 105 1B010 EVQLVESGGGLVQPGGSLTLSCVASGSTSRINAMGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTKSGSSMRYWGQGTLVTVSS 106 1B011 EVQLVESGGGLVQPGGSLTLSCVASGSTSRINAMGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVYGSSMRYWGQGTLVTVSS 107 1C01 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGSKVYRDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMGYWGQGTLVTVSS Petition 870260057806, dated 06 / 15 / 2026, page 179 / 374 169 / 256 SEQ. ID NO. nome sequência 108 1C02 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYSDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRSWGQGTLVTVSS 109 1C03 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDNSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVGGSSMRYWGQGTLVTVSS 110 1C04 EVQLVESGGGLVQPGGSLTLSCVASGNTSSINAMAWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRYWGQGTLVTVSS 111 1C05 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSHMRYWGQGTLVTVSS 112 1C06 EVQLVESGGGLVQPGGSLTLSCVASGSTSIINAMGWYRRAPGKQREL VAGISSDGSKVYEDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRAVSGSSMRYWGQGTLVTVSS 113 1C07 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRYWGQGTLVTVSS 114 1C08 EVQLVESGGGLVQPGGSLTLSCVASGSTSRINAMGWYRRAPGKQREL PAGISSDGSKVYAVSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSPMRYWGQGTLVTVSS 115 1C010EVQLVESGGGLVQPGGSLTLSCVASGSTSRINAMGWYRRAPGKQREL VAGVSSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMSYWGQGTLVTVSS 116 1C011 EVQLVESGGGLVQPGGSLTLSCVASGTTSSINAMGWYRRAPGKQREL VAGISSDGSKVYEDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRYWGQGTLVTVSS 117 1C012 EVQLVESGGGLVQPGGSLTLSCVASGITSSINAMGWYRRAPGKQREL VAGISSDGSKVYAGSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVRGSSMRYWGQGTLVTVSS 118 1D01 EVQLVESGGGLVQPGGSLTLSCVASGSTSDINAMGWYRRAPGKQREL VAGISSDKSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVRGSSMRYWGQGTLVTVSS Petition 870260057806, dated 06 / 15 / 2026, pp. 180 / 374 170 / 256 SEQ. ID NO. nome sequência 119 1D02 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSNGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRQVSGSSMRYWGQGTLVTVSS 120 1D03 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAIGWYRRAPGKQREL VAGISSDGSKVLADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRIVSGSSMGYWGQGTLVTVSS 121 1D04 EVQLVESGGGLVQPGGSLTLSCVASGSTSSKNAMGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGASMRYWGQGTLVTVSS 122 1D06 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDNSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVHGSSMRYWGQGTLVTVSS 123 1D08 EVQLVESGGGLVQPGGSLTLSCVASGLTSSINAMGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRMVSGSSMRYWGQGTLVTVSS 124 1D09 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYTDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTISGSSMRYWGQGTLVTVSS 125 1D010 EVQLVESGGGLVQPGGSLTLSCVASGSTSSNNAMAWYRRAPGKQREL VAGISSDGSKVYTDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTRSGSSMRYWGQGTLVTVSS 126 1D011EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDNSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGHSMRYWGQGTLVTVSS 127 1D012 EVQLVESGGGLVQPGGSLTLSCVASGSTSHINAMGWYRRAPGKQREL VAGISSDGSRVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGGSMRYWGQGTLVTVSS 128 1E02 EVQLVESGGGLVQPGGSLTLSCVASGQTSSINAMGWYRRAPGKQREL VAGISSDGSQVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTKSGSSMRYWGQGTLVTVSS 129 1E04 EVQLVESGGGLVQPGGSLTLSCVASGSTSRINGMGWYRRAPGKQREL PAGISSDGSKAYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTASGTSMRYWGQGTLVTVSS Petition 870260057806, dated 06 / 15 / 2026, pp. 181 / 374 171 / 256 SEQ. ID NO. name sequence 130 1E05 EVQLVESGGGLVQPGGSLTLSCVASGSTSVINAMAWYRRAPGKQREL AAGISSDGSKVYAKSAKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFNTVSGSSMRYWGQGTLVTVSS 131 1E07 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYNDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVRGSSQRYWGQGTLVTVSS 132 1E08 EVQLVESGGGLVQPGGSLTLSCVASGKTSSINAMGWYRRAPGKQREL VAGISSDGSKVIADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVLGSSMRYWGQGTLVTVSS 133 1E09 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYTDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTRSGSSMRYWGQGTLVTVSS 134 1E010 EVQLVESGGGLVQPGGSLTLSCVASGSVSSINAMGWYRRAPGKQREL VAGISSDGSKVYIDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGLSMRYWGQGTLVTVSS 135 1E011 EVQLVESGGGLVQPGGSLTLSCVASGNTSSINAMGWYRRAPGKQREL VAGISSDGSKVYYDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVRGSSQRYWGQGTLVTVSS 136 1E012 EVQLVESGGGLVQPGGSLTLSCVASGSTSSTNAMGWYRRAPGKQREL VAGISSDGSKVYVDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMVYWGQGTLVTVSS 137 1F01EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGSKVYGDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSRSSMRYWGQGTLVTVSS 138 1F02 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL AAGISSDQSKVYADSAKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMSYWGQGTLVTVSS 139 1F04 EVQLVESGGGLVQPGGSLTLSCVASGGTSSINAMGWYRRAPGKQREL VAGISSDGSKVYSDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSARYWGQGTLVTVSS 140 1F05 EVQLVESGGGLVQPGGSLTLSCVASGSTRSINAMGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFHTVSGSSMRYWGQGTLVTVSS Petition 870260057806, dated 06 / 15 / 2026, page 182 / 374 172 / 256 SEQ. ID NO. nome sequência 141 1F06 EVQLVESGGGLVQPGGSLTLSCVASGTTSSINAMGWYRRAPGKQREL VAGISSDGSKVIADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVLGSSMRYWGQGTLVTVSS 142 1F07 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGSKVDADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRYWGQGTLVTVSS 143 1F08 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYKDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRNVSGSSMRYWGQGTLVTVSS 144 1F09 EVQLVESGGGLVQPGGSLTLSCVASGNTSSINAMGWYRRAPGKQREL VAGISSNGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVTGSSMRYWGQGTLVTVSS 145 1F010 EVQLVESGGGLVQPGGSLTLSCVASGSTSRINAMGWYRRAPGKQREL VAGISSDGSKVYKDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRYWGQGTLVTVSS 146 1F011 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAIGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVKGSSMRYWGQGTLVTVSS 147 1F012 EVQLVESGGGLVQPGGSLTLSCVASGLTSSINAMGWYRRAPGKQREL VAGISSDGSKVYQDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTNSGSSMRYWGQGTLVTVSS 148 1G01EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYAESVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGASMRYWGQGTLVTVSS 149 1G04 EVQLVESGGGLVQPGGSLTLSCVASGSTSSTNAMGWYRRAPGKQREL VAGISSDGSKVLADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVNLSSMRYWGQGTLVTVSS 150 1G05 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAIGWYRRAPGKQREL VAGISSDGSKYYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVTGSSMRYWGQGTLVTVSS 151 1G06 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAIGWYRRAPGKQREL VAGISSDGSKVYAVSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRKVSGSSARYWGQGTLVTVSS Petition 870260057806, dated 06 / 15 / 2026, pp. 183 / 374 173 / 256 SEQ. ID NO. nome sequência 152 1G07 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGS KVVAD SVKGRFTISRDNAKNSVYL QMN S LRAE DTAVYY CYYFRTYSGSSMRYWGQGTLVTVSS 153 1G09 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSKSSMRYWGQGTLVTVSS 154 1G011 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFKTVSGSSMRYWGQGTLVTVSS 155 1H01 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL AAGISSDNSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTRSGSSMRYWGQGTLVTVSS 156 1H02 EVQLVESGGGLVQPGGSLTLSCVASGSKSSINAMGWYRRAPGKQREL AAGISSDGSKVYAQSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTSSGSSMRYWGQGTLVTVSS 157 1H06 EVQLVESGGGLVQPGGSLTLSCVASGTTSSINAMGWYRRAPGKQREL VAGISSDGSKVYVDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRFLSGSSMRYWGQGTLVTVSS 158 1H07 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAFGWYRRAPGKQREL VAGISSDGSKVYSDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRYWGQGTLVTVSS 159 1H08EVQLVESGGGLVQPGGSLTLSCVASGSTFSINAMGWYRRAPGKQREL VAGISSDGSKVLADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRLVSGSSMRYWGQGTLVTVSS 160 1H010 EVQLVESGGGLVQPGGSLTLSCVASGSTRSINAMGWYRRAPGKQREL VAGISSDGSKVYNDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRFWGQGTLVTVSS 161 1H011 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAIGWYRRAPGKQREL VAGISSDGSKVYNDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTQSGSSMRYWGQGTLVTVSS 162 1H012 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGSKVYVDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMPYWGQGTLVTVSS Petition 870260057806, dated 06 / 15 / 2026, pp. 184 / 374 174 / 256 SEQ. ID NO. nome sequência 163 2A01 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGS KVVAD SVKGRFTISRDNAKNSVYL QMN S LRAE DTAVYY CYYFRTLSGSSMRYWGQGTLVTVSS 164 2A03 EVQLVESGGGLVQPGGSLTLSCVASGTTSSINAMGWYRRAPGKQREL VAGISSDGSKVYGDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSAMRYWGQGTLVTVSS 165 2A04 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYTDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTTSGSSMRYWGQGTLVTVSS 166 2A05 EVQLVESGGGLVQPGGSLTLSCVASGRTSSINAMGWYRRAPGKQREL VAGISSDGSKVYNDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGTSMRYWGQGTLVTVSS 167 2A0 6 EVQLVESGGGLVQPGGSLTLSCVASGSTSSRNAMGWYRRAPGKQREL VAGISSDGSKVTADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTRSGSSMRYWGQGTLVTVSS 168 2A08 EVQLVESGGGLVQPGGSLTLSCVASGSTKSINAMGWYRRAPGKQREL VAGISSDGSKVYRDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTSSGSSMRYWGQGTLVTVSS 169 2A0 9 EVQLVESGGGLVQPGGSLTLSCVASGSTSSRNAMGWYRRAPGKQREL VAGISSNGSKVYSDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMSYWGQGTLVTVSS 170 2A011EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAIGWYRRAPGKQREL VAGISSDGSKVYSDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRPVSGSSMRYWGQGTLVTVSS 171 2B01 EVQLVESGGGLVQPGGSLTLSCVASGSTSLINAMGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRHVSGSSMRYWGQGTLVTVSS 172 2B02 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTKSGSSMRYWGQGTLVTVSS 173 2B03 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMGWYRRAPGKQREL VAGISSDGSLVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFTTVSGSSMRYWGQGTLVTVSS Petição 870260057806, de 15 / 06 / 2026, pág. 185 / 374 175 / 256 SEQ. ID NO. nome sequência 174 2B05 EVQLVESGGGLVQPGGSLTLSCVASGTTSSINAMGWYRRAPGKQREL VAGISSDGTKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFHTVSGSSMRYWGQGTLVTVSS 175 2B07 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAFGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVRGSSMRYWGQGTLVTVSS 176 2B010 EVQLVESGGGLVQPGGSLTLSCVASGSTSSRNAMGWYRRAPGKQREL VAGISSDGSKLYLDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVLGSSMRYWGQGTLVTVSS 177 2B011 EVQLVESGGGLVQPGGSLTLSCVASGNTSSINAMGWYRRAPGKQREL VAGISSDGSRVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVSGSSMRSWGQGTLVTVSS 178 2B012 EVQLVESGGGLVQPGGSLTLSCVASGTTSSINAMGWYRRAPGKQREL VAGISSDGSKVYNDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVRGSSMRYWGQGTLVTVSS 179 2C01 EVQLVESGGGLVQPGGSLTLSCVASGSTASINAMGWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRYVSGSSMRYWGQGTLVTVSS 180 2C02 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAVGWYRRAPGKQREL VAGISSDGSKVYVDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVYGSSMRYWGQGTLVTVSS 181 2C04EVQLVESGGGLVQPGGSLTLSCVASGSTSSRNAMGWYRRAPGKQREL VAGISSDGSKLYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRTVLGSSMRYWGQGTLVTVSS 182 2C06 EVQLVESGGGLVQPGGSLTLSCVASGSTNSINAMGWYRRAPGKQREL VAGISSDGSKVYKDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYYRTVSGSSMRYWGQGTLVTVSS 183 2C07 EVQLVESGGGLVQPGGSLTLSCVASGSTSSINAMAWYRRAPGKQREL VAGISSDGSKVYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRSVSGSSMRYWGQGTLVTVSS 184 2C08 EVQLVESGGGLVQPGGSLTLSCVASGSTSRINAMGWYRRAPGKQREL VAGISSDGSKVYQDSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYY CYYFRRVSGSSMRYWGQGTLVTVSS Petition 870260057806, dated 06 / 15 / 2026, pp. 186 / 374 176 / 256 SEQ. ID NO. nome sequência 185 2C09 EVQLVESGGGLVQPGGSLTLSCVPSGSTSNINAMGWYRRAPGKQREL PAGISSDGTKIYADSAKVPFTITRDNAKNSVYLQMNSL...

Claims

CLAIMS 1. Trispecific protein targeting DLL3, said protein being characterized by comprising: (a) a first domain (A) that specifically binds to human CD3; (b) a second domain (B) that is a single-domain antibody that specifically binds to a human serum albumin protein; and (c) a third domain (C) that specifically binds to a DLL3 protein, wherein the third domain (C) is a single-domain antibody comprising a CDR1, a CDR2 and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 851, 867, 871, 872, 873 and 1887; the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 1293, 1309, 1313, 1314, 1315 and 1888; and a CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 1735, 1751, 1755, 1756, 1757 and 1889.

2. Trispecific protein targeting DLL3, according to claim 1, characterized in that the domains are linked in the order of H2N-(A)-(B)-(C)-COOH, H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(B)-(C)-(A)-COOH, H2N-(C)-(B)-(A)COOH or H2N-(C)-(A)-(B)-COOH, or by L1 and L2 ligands, in the order H2N-(A)-L1-(B)-L2-(C)-COOH, H2N-(A)-L1-(C)-L2-(B)-COOH, H2N(B)-L1-(A)-L2-(C)-COOH, H2N-(B)-L1-(C)-L2-(A)-COOH, H2N-(C)-L1(B)-L2-(A)-COOH or H2N-(C)-L1-(A)-L2-(B)-COOH.

3. Trispecific protein targeting DLL3, according to claim 2, characterized in that the domains are linked in the order H2N-(A)-(B)-(C)-COOH or by L1 and L2 linkers in the order H2N-(A)-L1-(B)-L2-(C)-COOH.

4. Trispecific protein targeting DLL3 according to claim 3, characterized in that the L1 and L2 ligands independently comprise the sequence GGGGSGGGS (SEQ ID NO: 1808). Petition 870260081775, dated 08 / 13 / 2026, p. 11 / 38 2 / 4 5. Trispecific protein targeting DLL3, according to any one of claims 1 to 4, characterized in that the third domain (C) comprises a sequence that is at least about 80% identical to a sequence selected from the group consisting of SEQ ID NOS. 408, 425, 429, 430, 431 and 1886.

6. Trispecific protein targeting DLL3, according to any one of claims 1 to 5, characterized in that the third domain (C) comprises the sequences SEQ ID NOS. 408, 425, 429, 430, 431 or 1886.

7. Trispecific protein targeting DLL3, according to any one of claims 1 to 6, characterized in that the second domain (B) comprises a sequence selected from the group consisting of SEQ ID NOS. 1769-1778.

8. Trispecific protein targeting DLL3, according to any one of claims 1 to 7, characterized in that the first domain (A) comprises a sequence selected from the group consisting of SEQ ID NOS. 17931802 and 1897-1898.

9. Trispecific protein targeting DLL3, according to any one of claims 1 to 6, characterized in that: (a) the first domain (A) comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH comprises an HCDR1 comprising sequence SEQ ID NO: 1830, an HCDR2 comprising sequence SEQ ID NO: 1840 and an HCDR3 comprising sequence SEQ ID NO: 1842; and wherein the VL comprises an LCDR1 comprising sequence SEQ ID NO: 1852, an LCDR2 comprising sequence SEQ ID NO: 1877 and an LCDR3 comprising sequence SEQ ID NO: 1878; and / or; (b) the second domain (B) comprises a CDR1 comprising the sequence SEQ ID NO: 1783, a CDR2 comprising the sequence SEQ ID NO: 1787 and a CDR3 comprising the sequence SEQ ID NO: Petition 870260081775, dated 13 / 08 / 2026, page 12 / 38 3 / 4 1792.

10. Trispecific protein targeting DLL3, according to claim 9, characterized in that: (a) the first domain (A) comprises the sequence SEQ ID NO: 1793; and / or (b) the second domain (B) comprises the sequence SEQ ID NO: 1774.

11. Polynucleotide, characterized by encoding the trispecific protein that targets DLL3, as defined in any one of claims 1 to 10.

12. Vector, characterized by comprising the polynucleotide, as defined in claim 11.

13. Host cell, characterized by comprising the polynucleotide as defined in claim 11 or the vector as defined in claim 12.

14. Use of the trispecific protein targeting DLL3, as defined in any one of claims 1 to 10, characterized in that it is in the manufacture of a medicament for the treatment of a cancer that expresses DLL3.

15. Use, according to claim 14, characterized in that the cancer comprises lung cancer, neuroendocrine cancer, gastric cancer, ovarian cancer, or triple-negative breast cancer.

16. Use according to claim 15, characterized in that (i) the lung cancer comprises small cell lung cancer; or (ii) the neuroendocrine cancer comprises neuroendocrine prostate cancer.

17. Pharmaceutical composition, characterized by comprising: (i) the trispecific protein targeting DLL3, as defined in any one of claims 1 to 10, the polynucleotide, as defined in claim 11, or the vector, as defined in claim 12, and (ii) a pharmaceutically acceptable carrier. Petition 870260081775, dated 13 / 08 / 2026, page 13 / 38 4 / 4 18. Method for preparing a trispecific DLL3-targeting protein, as defined in any one of claims 1 to 10, characterized by 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 from the library a DLL3-binding protein that specifically binds to an oligomer or derivative thereof; and iv) preparing the trispecific DLL3-targeting protein using the DLL3-binding protein identified in step (iii).

19. Process for the production of a trispecific protein targeting DLL3, said process being characterized by comprising cultivating a transformed or transfected host with a vector comprising the nucleic acid sequence encoding the trispecific protein targeting DLL3, as defined in any one of claims 1 to 10, under conditions that permit the expression of the trispecific protein targeting DLL3 and recovering and purifying the protein produced from the culture.

20. Trispecific protein targeting DLL3, characterized by being produced by the process as defined in claim 19.

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