Fusion molecule of immune cell engager and cytokine

Abstract

Fusion molecules comprising an immune cell engager and a cytokine are disclosed. Also disclosed are nucleic acids and expression vectors encoding, compositions comprising, and methods of using, the fusion molecules.

Description

[0001] Immune Cell Engagers

[0002] This application claims priority from US 63 / 729,104 filed 6 December 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present disclosure relates to the fields of molecular biology, more specifically immune cell engager technology. The present invention also relates to methods of medical treatment and prophylaxis.

[0005] Background

[0006] Immune cell engager molecules (such as T cell engagers (TCEs) and Bi-specific T cell engagers (BiTE) are a class of immunotherapies that recruit / engage immune cells and direct effector activity against cells expressing a target antigen. They comprise an antigen-binding region for a target antigen of interest (e.g. a tumor antigen), and an antigen-binding region specific for an immune cell (e.g. an immune cell surface molecule).

[0007] Following the FDA's approval of TCEs for treating B cell malignancies and solid tumors (see e.g. Przepiorka et al., Clin Cancer Res. (2015) 15;21 (18):4035-9, Hua et al., J Adv Pract Oncol. (2023) 14(2):163-171 and Giffin et al., (2021) Clin Cancer Res. 27(5):1526-1537), there has been a surge in innovative immune cell engager designs, including bi-specific natural killer (NK) cell engagers (BiKEs), bispecific macrophage engagers (BiMEs), and dendritic cell engagers. For example, see Gleason et al., Blood. (2014) 8;123(19):3016-26, Tapia-Galisteo et al., J Hematol Oncol. (2023) 27;16(1 ):83, et al. Cell. (2024) 18;187(2):375-389.e18 and Sun et al; Cancer Res (2024); 84 (6_Supplement):183, which are all hereby incorporated by reference in their entirety. While these advances are promising, there remains challenges and rooms for improvement in therapeutic outcomes. For example, the tumor microenvironment (TME) poses several challenges for T cell therapies, including its immunosuppressive nature, limited immune cell infiltration, and the development of T cell exhaustion. T cell exhaustion, a state where T cells lose their ability to proliferate and survive due to persistent antigen stimulation, significantly reduces the effectiveness of TCEs in controlling tumor growth. See e.g. Tie et al. (2022) (J Hematol Oncol. 18;15(1):61), Chow et al. (2022) (Nat Rev Clin Oncol. 19(12)775-790), Jiang et al., (2015) (Cell Death Dis. 6(6):e1792) and Jia et al., (2023) (Front Immunol. 14:1303605), which are all hereby incorporated by reference in their entirety.

[0008] To tackle complex diseases such as cancer, it is crucial to adopt multiple strategies that target different components of the interconnected regulatory networks. However, administering combinations of individual drugs introduces challenges such as production complexities, regulatory obstacles, and safety concerns (see e.g. Humphrey et al. J Natl Cancer Inst. (2011) 17; 103(16): 1222-6 and Lopez et al. Nat Rev Clin Oncol. (2017) 14(1 ):57-66, which are both hereby incorporated by reference in their entirety). Multifunctional proteins offer a more streamlined solution by combining multiple therapeutic functions within a single construct. Despite this, they present unique challenges. The engineering of multifunctional proteins is highly intricate and logistically demanding. The present invention has been devised in light of the above considerations. The present invention also aims to address the problem that patients with variable immune cells can lead to poor responses using current immune cell engagers.

[0009] Summary

[0010] In a first aspect, the present disclosure provides a fusion molecule comprising an immune cell engager and a cytokine.

[0011] In some embodiments, the immune cell engager comprises an immune cell-binding moiety, a tumor antigen-binding moiety and an Fc region.

[0012] In some embodiments, the fusion molecule is a polypeptide complex comprising, or consisting of:

[0013] (i) a first polypeptide comprising a tumor antigen-binding moiety, an immune cell-binding moiety, and a first CH2-CH3 region; and

[0014] (ii) a second polypeptide comprising a second CH2-CH3 region and a cytokine.

[0015] In some embodiments, the fusion molecule is a polypeptide complex comprising or consisting of:

[0016] (i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, an immune cell-binding moiety, and a first CH2-CH3 region; and

[0017] (ii) a second polypeptide comprising from N to C terminus: a second CH2-CH3 region and a cytokine.

[0018] In some embodiments, the fusion molecule is a polypeptide complex comprising or consisting of:

[0019] (i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, a first CH2-CH3 region, and an immune cell-binding moiety; and

[0020] (ii) a second polypeptide comprising from N to C terminus: a second CH2-CH3 region and a cytokine.

[0021] In some embodiments, the second polypeptide comprises a linker between the second CH2-CH3 region and the cytokine, optionally wherein the linker comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 29, 31 , 32, 68, 69, 70, 71 , 72, 73, 90 or 91 .

[0022] In some embodiments, the Fc region comprises at least one modification to promote heterodimerization, reduce antibody-dependent cellular cytotoxicity, and / or extend the serum half-life. In some embodiments, the Fc region comprises knob-in-hole modifications.

[0023] In some embodiments, the cytokine is IL-15, IL-2, IL-4, IL-7, IL-9, IL-12, IL-18, IL-10, or IL-21 , or is an engineered cytokine.

[0024] In some embodiments, the cytokine is an engineered cytokine. In some embodiments, the cytokine is sushi-IL-15, IL-15-sushi or Neo-2 / 15. In some embodiments, the cytokine is IL-15, or an IL-15 / sushi domain fusion construct.

[0025] In some embodiments, the immune cell-binding moiety is a T-cell binding moiety.

[0026] In some embodiments, the immune cell-binding moiety is a scFv, VH domain, Fab or nanobody.

[0027] In some embodiments, the immune cell-binding moiety specifically binds CD2, CD3, CD16, CD28, CD54, CD137 or CD335. In some embodiments, the immune cell-binding moiety specifically binds CD3.

[0028] In some embodiments, the tumor antigen-binding moiety is a scFv, VH domain, Fab or nanobody.

[0029] In some embodiments, the tumor antigen-binding moiety specifically binds a cancer cell antigen or a cancer-associated antigen. In some embodiments, the tumor antigen-binding moiety specifically binds an antigen selected from DLL3, EpCAM, TROP2, SEZ6, KLK2, PSMA, STEAP1 , STEAP2, CLDN6 and MUC16. In some embodiments, the tumor antigen-binding moiety specifically binds DLL3.

[0030] In a further aspect is provided a polynucleotide or plurality of polynucleotides encoding the fusion molecules of the first aspect comprising an immune cell engager and a cytokine.

[0031] In a further aspect, the present disclosure provides a pharmaceutical composition comprising a fusion molecule comprising an immune cell engager and a cytokine, and a pharmaceutically acceptable carrier.

[0032] In a further aspect, the present disclosure provides a fusion molecule comprising an immune cell engager and a cytokine, a polynucleotide encoding a fusion molecule described herein, or a pharmaceutical composition comprising a fusion molecule described herein and a pharmaceutically acceptable carrier, for use in a method of treating or preventing a disease or condition.

[0033] In a further aspect, the present disclosure provides a method of treating or preventing a disease or condition, comprising administering a fusion molecule comprising an immune cell engager and a cytokine, a polynucleotide encoding a fusion molecule described herein, or a pharmaceutical composition comprising a fusion molecule described herein and a pharmaceutically acceptable carrier, to a patient in need thereof.

[0034] In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is small cell lung cancer, non-small cell lung cancer, neuroendocrine cancer (e.g., NEPC, LCNEC, NEBC), prostate cancers, breast cancers, gastric cancer, pancreatic cancer, liver cancer, lymphoma, leukaemia, myeloma, brain tumors, skin cancer, uterine cancer, and kidney cancer.

[0035] In a further aspect, the present disclosure provides a method of treating cancer, comprising administering a fusion molecule comprising an immune cell-binding moiety, a tumor antigen-binding moiety, an Fc region and a cytokine. In a further aspect, the present disclosure provides a fusion molecule comprising an immune cell-binding moiety, a tumor antigen-binding moiety, an Fc region and a cytokine for use in a method of treating cancer.

[0036] Description

[0037] Immune cell engagers comprise an antigen-binding region for a target antigen of interest (a tumor antigen-binding moiety), and an antigen-binding region for recruiting / engaging an immune cell of interest (an immune cell-binding moiety). Immune cell engagers are reviewed e.g. in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17: 418-434 and Ellerman, Methods (2019) 154:102-117, both of which are hereby incorporated by reference in their entirety.

[0038] This invention addresses the clinical issue that current immune-cell recruiters exhaust T cells through stimulation by providing fusion molecules comprising an immune cell engager and a cytokine. In all embodiments described herein, the fusion molecule may be a fusion protein.

[0039] Disclosed herein is a fusion molecule comprising an immune cell engager and a cytokine, wherein the immune cell engager comprises an immune cell-binding moiety, a tumor antigen-binding moiety and an Fc region. Accordingly, disclosed herein is a fusion molecule comprising an immune cell-binding moiety, a tumor antigen-binding moiety, an Fc region and a cytokine.

[0040] The best studied immune cell engagers are bispecific T cell engagers (BiTEs). Other kinds of immune cell engagers include natural killer cell engagers such as bispecific killer engagers (BiKEs), which recruit and activate NK cells. In some embodiments, the immune cell engaged by the immune cell engager is a T cell, NK cell, macrophage, or a dendritic cell.

[0041] In some embodiments, the immune cell engager is a T cell-engager. Canonical BiTEs consist of two single-chain variable fragments (scFvs) of different antibodies; a target antigen binding domain (which binds a tumor antigen on a cancer cell), and a CD3 polypeptide (typically CD3e)-binding domain, through which the BiTE recruits T cells. Binding of the BiTE to its target antigen and to the CD3 polypeptide expressed by the T cell results in activation of the T cell, and ultimately directs T cell effector activity against cells expressing the target antigen.

[0042] Several half-life-extended (HLE) BiTE molecules (a canonical BiTE molecule fused to an Fc domain) are in development. For example, Tarlatamab (also known as AMG 757; DrugBank Acc. No: DB17256) is a HLE BiTE consisting of an anti-DLL3 scFv, an anti-CD3e scFv and an Fc domain) as described in Giffin et al., (2021) Clin Cancer Res. 27(5):1526-1537. HLE BiTEs are reviewed in Einsele et al., (2020) (Cancer. 126(14):3192-3201), which is hereby incorporated by reference in its entirety.

[0043] In some embodiments, the immune cell engager is a NK cell-engager. NK cell-engagers (NKCEs) are reviewed in Zhang et al., (2023) (Front Immunol. 14:1207276, which is hereby incorporated by reference in its entirety) and include BiKEs and TriKEs (trispecific killer cell engagers). Immune Cell Bindinq-Moieties

[0044] The fusion molecules of the present disclosure comprise an immune cell-binding moiety. An immune cellbinding moiety is a moiety (e.g. an antigen-binding moiety) that selectively binds to an immune cell. For example, an immune cell-binding moiety may selectively bind an immune cell surface molecule. An immune cell surface molecule may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed at or on the cell surface of an immune cell. In some embodiments, the part of the immune cell surface molecule which is bound by the fusion molecule of the present disclosure is on the external surface of the immune cell ( / .e. is extracellular). The immune cell surface molecule may be expressed at the cell surface of any immune cell.

[0045] In some embodiments, the immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, macrophage or monocyte. The lymphocyte may be e.g. a T cell, B cell, natural killer (NK) cell, NKT cell, y5T cell or innate lymphoid cell (ILC), or a precursor thereof (e.g. a thymocyte or pre-B cell). In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a NK cell.

[0046] In some embodiments, the immune cell-binding moiety specifically binds CD2, CD3, CD16, CD28, CD54, CD137 or CD335. In some embodiments, the immune cell-binding moiety is a scFv, VH domain, Fab, or nanobody which specifically binds CD2, CD3, CD16, CD28, CD54, CD137 or CD335.

[0047] In some embodiments, the immune cell-binding moiety is a T-cell binding moiety. In such embodiments, the immune cell-binding moiety binds a target on (e.g. expressed on the surface of) a T cell, for example CD3, the invariable part of the T-cell receptor complex. In some embodiments, the immune cell-binding moiety specifically binds CD3 (in other words, the immune cell-binding moiety is a CD3-binding moiety), for example, a CD3 polypeptide (e.g. CD3e, CD36, CD3y or CD3Q. In some embodiments, the immune cell-binding moiety is a scFv, VH domain, Fab, or nanobody which specifically binds CD3. In some embodiments, the immune-cell binding moiety is a scFv which specifically binds to CD3.

[0048] In some embodiments, the immune cell-binding moiety according to the present disclosure comprises the heavy chain CDR sequences according to SEQ ID NO:62, 63 and 64, and the light chain CDR sequences according to SEQ ID NO:65, 66 and 67.

[0049] In some embodiments, the immune cell-binding moiety according to the present disclosure comprises an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 60. In some embodiments, the immune cell-binding moiety according to the present disclosure comprises an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 61 . In some embodiments, the immune cell-binding moiety according to the present disclosure comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 1 .

[0050] In some embodiments, the immune cell-binding moiety according to the present disclosure comprises the heavy chain CDR sequences according to SEQ ID NO:54, 55 and 56, and the light chain CDR sequences according to SEQ ID NO:57, 58 and 59.

[0051] In some embodiments, the immune cell-binding moiety according to the present disclosure comprises an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 52. In some embodiments, the immune cell-binding moiety according to the present disclosure comprises an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 53.

[0052] In some embodiments, the immune cell-binding moiety according to the present disclosure comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 51 .

[0053] In some embodiments, the immune cell-binding moiety is a NK-cell binding moiety. In such embodiments, the immune cell-binding moiety binds a target on (e.g. expressed on the surface of) a NK cell, for example CD16. In some embodiments, the immune cell-binding moiety specifically binds CD16, for example CD16a. In some embodiments, the immune cell-binding moiety is a scFv which specifically binds CD16.

[0054] Tumor Antigen-Binding Moieties

[0055] The fusion molecules of the present disclosure comprise a tumor antigen-binding moiety. A tumor antigen-binding moiety is a moiety that selectively binds to a tumor antigen. A tumor antigen is an antigen which is expressed or over-expressed by a tumor.

[0056] In some embodiments, the tumor antigen-binding moiety may bind a cancer cell antigen. A cancer cell antigen is an antigen which is expressed or over-expressed by a cancer cell. A cancer cell antigen may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. A cancer cell antigen’s expression may be associated with a cancer. A cancer cell antigen may be abnormally expressed by a cancer cell (e.g. the cancer cell antigen may be expressed with abnormal localisation), or may be expressed with an abnormal structure by a cancer cell. A cancer cell antigen may be capable of eliciting an immune response. In some embodiments, the antigen is expressed at the cell surface of the cancer cell ( / .e. the tumor antigen is a cancer cell surface antigen). In some embodiments, the part of the antigen which is bound by the antigen-binding molecule described herein is displayed on the external surface of the cancer cell ( / .e. is extracellular).

[0057] The cancer cell antigen may be a cancer-associated antigen. Accordingly, in some embodiments, the tumor antigen-binding moiety may bind a cancer-associated antigen. In some embodiments the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer. The cancer-associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer. In some embodiments, the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and / or protein level) by cells of a cancer, e.g. as compared to the level of expression by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue / cell type). In some embodiments, the cancer-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue / cell type). In some embodiments, the cancer-associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene. In some embodiments, the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.

[0058] Cancer-associated antigens are reviewed by Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003. Cancer-associated antigens include oncofetal antigens: CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: fibroblast activation protein (FAP), B-cell maturation antigen (BCMA), CD19, HER2 / neu, HER3, BING-4, calcium-activated chloride channel 2, cyclin-B1 , 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP- 1 , survivin, delta-like ligand 3 (DLL3), tumor-associated calcium signal transducer 2 (TROP2), epidermal growth factor receptor (EGFR), mesenchymal-epithelial transition factor (cMET), B7-H3, B7-H4, PKMYT1 , CEACAM-5, Fra, CLL-1 , GPC3, Six transmembrane epithelial antigen of the prostate 1 (STEAP1), Six transmembrane epithelial antigen of the prostate 2 (STEAP2), Mucin 1 (MUC1), Mucin 16 (MUC16), Claudin family proteins (e.g. CLDN6, CLDN18.2), SEZ6, KLK2, Mesothelin, CD70, EphAIO, CTGF, Nectin-4, AXL, Liv-1 , SLC34A2; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1 , PRAME, SSX-2; lineage restricted antigens: MARTI , Gp100, tyrosinase, TRP-1 / 2, MC1 R, prostate specific antigen prostate-specific membrane antigen (PSMA); mutated antigens: p-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, MART-2, p53, Ras, TGF-pRII, FGFR1 , FGFR2, FGFR3, FGFR4; post-translationally altered antigens: MUC1 , idiotypic antigens: Ig, TCR. Other cancer cell antigens include heat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose- regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B. In some embodiments the cancer cell antigen is a cancer cell antigen described in Zhao and Cao, Front Immunol. (2019) 10:2250, which is hereby incorporated by reference in its entirety. In some embodiments, the tumor antigen-binding moiety specifically binds an antigen selected from: CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: fibroblast activation protein (FAP), B-cell maturation antigen (BCMA), CD19, HER2 / neu, HER3, BING-4, calcium-activated chloride channel 2, cyclin-B1 , 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP-1 , survivin, delta-like ligand 3 (DLL3), tumor-associated calcium signal transducer 2 (TROP2), epidermal growth factor receptor (EGFR), mesenchymal-epithelial transition factor (cMET), B7-H3, B7- H4, PKMYT1 , CEACAM-5, Fra, CLL-1 , GPC3, Six transmembrane epithelial antigen of the prostate 1 (STEAP1), Six transmembrane epithelial antigen of the prostate 2 (STEAP2), Mucinl (MUC1), Mucin 16 (MUC16), Claudin family proteins (e.g. CLDN6, CLDN18.2), SEZ6, KLK2, Mesothelin, CD70, EphAIO, CTGF, Nectin-4, AXL, Liv-1 , SLC34A2; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1 , PRAME, SSX-2; lineage restricted antigens: MARTI , Gp100, tyrosinase, TRP-1 / 2, MC1 R, prostate specific antigen, prostate-specific membrane antigen (PSMA); mutated antigens: p-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, MART-2, p53, Ras, TGF-pRII, FGFR1 , FGFR2, FGFR3, FGFR4; post-translationally altered antigens: MUC1 , idiotypic antigens: Ig, TCR, heatshock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL- 2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B.

[0059] In some embodiments, the tumor antigen-binding moiety specifically binds an antigen selected from DLL3, EpCAM, TROP2, SEZ6, KLK2, PSMA, STEAP1 , STEAP2, CLDN6 and MUC16.

[0060] In some embodiments, the tumor antigen-binding moiety is a scFv, VH domain, Fab or nanobody which specifically binds an antigen selected from CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: fibroblast activation protein (FAP), B-cell maturation antigen (BCMA), CD19, HER2 / neu, HER3, BING-4, calcium-activated chloride channel 2, cyclin-B1 , 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP-1 , survivin, delta-like ligand 3 (DLL3), tumor-associated calcium signal transducer 2 (TROP2), epidermal growth factor receptor (EGFR), mesenchymal-epithelial transition factor (cMET), B7-H3, B7-H4, PKMYT1 , CEACAM-5, Fra, CLL-1 , GPC3, Six transmembrane epithelial antigen of the prostate 1 (STEAP1), Six transmembrane epithelial antigen of the prostate 2 (STEAP2), Mucin 1 (MUC1), Mucin 16 (MUC16), Claudin family proteins (e.g. CLDN6, CLDN18.2), SEZ6, KLK2, Mesothelin, CD70, EphAIO, CTGF, Nectin-4, AXL, Liv-1 , SLC34A2; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1 , PRAME, SSX-2; lineage restricted antigens: MARTI , Gp100, tyrosinase, TRP-1 / 2, MC1 R, prostate specific antigen prostate-specific membrane antigen (PSMA); mutated antigens: p-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, MART- 2, p53, Ras, TGF-pRII, FGFR1 , FGFR2, FGFR3, FGFR4; post-translationally altered antigens: MUC1 , idiotypic antigens: Ig, TCR, heat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose- regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B. In some embodiments, the tumor antigen-binding moiety is a scFv, VH domain, Fab or nanobody which specifically binds an antigen selected from DLL3, EpCAM, TROP2, SEZ6, KLK2, PSMA, STEAP1 , STEAP2, CLDN6 and MUC16.

[0061] In some embodiments, the tumor antigen-binding moiety is a scFv which specifically binds DLL3. In some embodiments, the tumor antigen-binding moiety according to the present disclosure comprises the heavy chain CDR sequences according to SEQ ID NO:84, 85 and 86, and the light chain CDR sequences according to SEQ ID NO:87, 88 and 89. In some embodiments, the tumor antigen-binding moiety according to the present disclosure comprises an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 82. In some embodiments, the tumor antigen-binding moiety according to the present disclosure comprises an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 83.

[0062] In some embodiments, the tumor antigen-binding moiety according to the present disclosure comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 2.

[0063] In some embodiments, the tumor antigen-binding moiety is a VH domain which specifically binds DLL3.

[0064] In some embodiments, the tumor antigen-binding moiety according to the present disclosure comprises the heavy chain CDR sequences according to SEQ ID NO:48, 49 and 50.

[0065] In some embodiments, the tumor antigen-binding moiety according to the present disclosure comprises an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO:47.

[0066] Antigen-Binding Molecules

[0067] An immune cell-binding moiety or tumor antigen-binding moiety according to the present disclosure may comprise, or consist of, an antigen-binding molecule.

[0068] By way of example, the immune cell-binding moiety may be an antigen-binding molecule which specifically binds CD3, and the tumor antigen-binding moiety may be an antigen-binding molecule which specifically binds a cancer cell antigen or cancer-associated antigen as described hereinabove.

[0069] An ‘antigen-binding molecule’ refers to a molecule that binds to a given antigen. Antigen-binding molecules include antigen-binding polypeptides and antigen-binding polypeptide complexes such as antibodies ( / .e. immunoglobulins (Igs)) and antigen-binding fragments thereof. In some embodiments, an immune cell-binding moiety or tumor antigen-binding moiety according to the present disclosure comprises, or consists of, an antigen-binding polypeptide or an antigen-binding polypeptide complex. A ‘polypeptide’ is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.

[0070] An antigen-binding molecule may comprise more than one polypeptide which together form an antigenbinding domain. The polypeptides may associate covalently or non-covalently. An antigen-binding molecule may refer to a non-covalent or covalent complex of more than one polypeptide (e.g. 2, 3, 4, 6, or 8 polypeptides), e.g. an IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides. In some embodiments, the polypeptides form part of a larger polypeptide comprising the polypeptides (e.g. in the case of scFv comprising VH and VL, or in the case of scFab comprising VH-CH1 and VL-CL). In some embodiments, the polypeptides form part of the VH domain or nanobody.

[0071] In some embodiments, an immune cell-binding moiety or tumor antigen-binding moiety according to the present disclosure comprises, or consists of, an antibody. As used herein, ‘antibodies’ include monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, and antibody-derived target-binding molecules such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (sdAbs) (e.g. VH and VHH), etc. Antigen-binding fragments of antibodies include e.g. Fv, Fab, F(ab’)2 and F(ab’) fragments. Antigen-binding molecules also include polypeptide aptamers, thioredoxins, monobodies, anticalins, Kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affibodies, nanobodies ( / .e. a single-domain antibodies (sdAbs)), affilins, armadillo repeat proteins (ArmRPs), OBodies and fibronectins - reviewed e.g. in Reverdatto et al., Curr Top Med Chem. (2015) 15(12): 1082— 1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem. (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0072] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1): D671-D674. The CDRs and FRs of the VH regions and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77. In preferred embodiments, the CDRs and FRs of antigenbinding molecules referred to herein are defined according to the IMGT information system.

[0073] In some embodiments, the immune cell-binding moiety or tumor antigen-binding moiety comprises, or consists of, a scFv, VH domain, Fab or nanobody.

[0074] In some embodiments, an immune cell-binding moiety or tumor antigen-binding moiety according to the present disclosure comprises, or consists of, an scFv. An scFv comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to a given target molecule. An antigen-binding molecule formed by the VH and VL of a given antibody may also be described as the Fv region of the given antibody.

[0075] In some embodiments, a fusion molecule as described herein may comprise (i) an immune cell-binding moiety that comprises, or consists of, an scFv, and (ii) a tumor antigen-binding moiety that comprises, or consists of, an scFv. Accordingly, in some embodiments a fusion molecule as described herein comprises multiple scFvs.

[0076] In some embodiments, an immune cell-binding moiety or tumor antigen-binding moiety according to the present disclosure comprises, or consists of, a single-domain antibody (sdAb). In some embodiments, the immune cell-binding moiety or tumor antigen-binding moiety comprises, or consists of, a VHH (also called a nanobody). In some embodiments, the immune cell-binding moiety or tumor antigen-binding moiety comprises, or consists of, a VH domain.

[0077] In some embodiments, a fusion molecule as described herein may comprise (i) an immune cell-binding moiety that comprises, or consists of, an scFv, and (ii) a tumor antigen-binding moiety that comprises, or consists of, a sdAb (e.g. a nanobody or VH domain).

[0078] In some embodiments, the immune cell-binding moiety or tumor antigen-binding moiety according to the present disclosure comprises, or consists of, the Fab region of an antibody (also called a Fab). The VL and light chain constant (CL) region, and the VH region and heavy chain constant 1 (CH1) region of an antigen-binding region of an antibody together constitute the Fab region. In some embodiments, the immune cell-binding moiety or tumor antigen-binding moiety comprises, or consists of, a Fab region comprising a VH, a CH1 , a VL and a CL. In some embodiments, the VH, CH1 , VL and CL regions of the Fab are provided as single polypeptide joined by linker regions, i.e. as a single chain Fab (scFab).

[0079] In some embodiments, a fusion molecule as described herein may comprise (i) an immune cell-binding moiety that comprises, or consists of, an scFv, and (ii) a tumor antigen-binding moiety that comprises, or consists of, an antigen-binding fragment (e.g. a Fab).

[0080] By way of example, the immune cell-binding moiety may be an scFv which specifically binds CD3. In some embodiments, a fusion molecule as described herein may comprise (i) an immune cell-binding moiety that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 1 , and (ii) a tumor antigen-binding moiety that comprises, or consists of, an scFv, VH domain, nanobody, or Fab.

[0081] In some embodiments, a fusion molecule as described herein may comprise (i) an immune cell-binding moiety that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 1 , and (ii) a tumor antigen-binding moiety that comprises, or consists of, an scFv, VH domain, nanobody, or Fab that specifically binds to DLL3. In some embodiments, a fusion molecule as described herein may comprise (i) an immune cell-binding moiety that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 1 , and (ii) a tumor antigen-binding moiety that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 2.

[0082] In some embodiments, the immune cell-binding moiety may be an scFv which specifically binds CD3 and the tumor antigen-binding moiety may be a VHH. In some embodiments, the immune cell-binding moiety may be an scFv which specifically binds CD3 and the tumor antigen-binding moiety may be a VHH that specifically binds to DLL3.

[0083] In some embodiments, a fusion molecule as described herein may comprise (i) an immune cell-binding moiety that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 1 , and (ii) a tumor antigen-binding moiety that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 47.

[0084] Fc Regions

[0085] Described herein are fusion molecules of the present disclosure comprising a half-life extending moiety. One approach to half-life extension is fusion of peptide(s) or polypeptide(s) e.g. one or more scFvs) to an Fc region.

[0086] Accordingly, disclosed herein are fusion molecules comprising an Fc region. As used herein, an ‘Fc region’ refers to a polypeptide complex formed by interaction between two polypeptides, each polypeptide comprising the CH2-CH3 region of an immunoglobulin (Ig) heavy chain constant sequence. In some embodiments, an Fc region is a single chain Fc (ScFc). The “Fc region” may also comprise the hinge region of an Ig heavy chain constant sequence.

[0087] Herein, a ‘CH2 region’ refers to an amino acid sequence corresponding to the CH2 region of an immunoglobulin (Ig). The CH2 region is the region of an Ig formed by positions 231 to 340 of the immunoglobulin constant region, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85. A ‘CH3 region’ refers to an amino acid sequence corresponding to the CH3 region of an immunoglobulin (Ig).

[0088] The CH3 region is the region of an Ig formed by positions 341 to 447 of the immunoglobulin constant region, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85. A ‘CH2-CH3 region’ refers to an amino acid sequence corresponding to the CH2 and CH3 regions of an immunoglobulin (Ig). The CH2-CH3 region is the region of an Ig formed by positions 231 to 447 of the immunoglobulin constant region, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85.

[0089] The hinge region may refer to the region of an Ig formed by positions 219 to 230 of the immunoglobulin constant region, according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85.

[0090] In some embodiments, the Fc region comprises at least one modification to promote heterodimerization. For example, in some embodiments the fusion molecule comprises an Fc region comprising modification in one or more of the CH2 and CH3 regions promoting association of the Fc region. Recombinant coexpression of constituent polypeptides of a fusion protein comprising an Fc region and subsequent association leads to several possible combinations. To improve the yield of the desired combinations of polypeptides in fusion molecules in recombinant production, it is advantageous to introduce in the Fc regions modification(s) promoting association of the desired combination of heavy chain polypeptides. Modifications may promote e.g. hydrophobic and / or electrostatic interaction between CH2 and / or CH3 regions of different polypeptide chains. Suitable modifications are described e.g. in Ha etal., Front Immnol. (2016) 7:394, which is hereby incorporated by reference in its entirety. Fc regions comprising such modifications may be called a “hetero Fc format” or a “heterodimeric Fc”.

[0091] Where an Fc region is described as comprising a modification, the modification may be present in one or both of the polypeptide chains which together form the Fc region.

[0092] In some embodiments, the fusion molecule of the present disclosure is provided with an Fc region comprising the ‘knob-into-hole’ or ‘KiH’ modification, e.g. as described e.g. in US 7,695,936 and Carter, J Immunol Meth. (2001) 248:7-15. Fc regions comprising such modifications may be called a “knob-into- hole Fc”. In such embodiments, one of the CH3 regions of the Fc region comprises a ‘knob’ modification, and the other CH3 region comprises a ‘hole’ modification. The ‘knob’ and ‘hole’ modifications are positioned within the respective CH3 regions so that the ‘knob’ can be positioned in the ‘hole’ in order to promote heterodimerisation (and inhibit homodimerisation) of the polypeptides and / or stabilise heterodimers. Knobs are constructed by substituting amino acids having small chains with those having larger side chains (e.g. tyrosine or tryptophan). Holes are created by substituting amino acids having large side chains with those having smaller side chains (e.g. alanine or threonine).

[0093] In some embodiments, one of the CH3 regions of an Fc region of the present disclosure comprises the substitution T366W (numbering of positions / substitutions in the Fc, CH2 and CH3 regions herein is according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991), and the other CH3 region of the Fc region comprises the substitution Y407V. In some embodiments, one of the CH3 regions of the Fc region comprises the substitution T366W, and the other CH3 region comprises the substitutions T366S and L368A. In some embodiments, one of the CH3 regions of the Fc region comprises the substitution T366W, and the other CH3 region comprises the substitutions Y407V, T366S and L368A.

[0094] The Fc region of any of the fusion molecules described herein may comprise knob-into-hole modifications. In some embodiments, one of the CH3 regions of the Fc region comprises the substitutions S354C and T366W (‘knob’ Fc), and the other CH3 region comprises the substitutions Y349C, Y407V, T366S and L368A (‘hole’ Fc).

[0095] Any of the fusion molecules described herein may comprise modifications to the Fc region to reduce or prevent an Fc-mediated function (e.g. antibody-dependent cellular cytotoxicity (ADCC) or complementdependent cytotoxicity (CDC)). In some embodiments, the Fc region comprises at least one modification to reduce or prevent ADCC. The ability of, and extent to which, a given antigen-binding molecule is able to induce ADCC of a given target cell type can be analysed e.g. according to the method described in Yamashita et al., Scientific Reports (2016) 6:19772 (hereby incorporated by reference in its entirety), or by 51 Cr release assay as described e.g. in Jedema et al., Blood (2004) 103: 2677-82 (hereby incorporated by reference in its entirety).

[0096] In some embodiments, the one or more modifications to reduce or prevent ADCC are a mutation at residue position L234A, L235A, or a combination thereof. In some embodiments the Fc region comprises a modification corresponding to the combination of substitutions L234A / L235A (LALA). In some embodiments, the Fc region comprises a modification corresponding to the combination of substitutions L234A / L235A / P329G (LALAPG). The LALAPG combination of substitutions has been shown to eliminate complement binding and ADCC (see Lo et al. J. Biol. Chem (2017) 292(9):3900-3908, hereby incorporated by reference in its entirety).

[0097] In some embodiments the Fc region comprises a CH2-CH3 region comprising modification corresponding to the combination of substitutions L234A / L235A (LALA) or L234A / L235A / P329G (LALAPG).

[0098] In some embodiments, the Fc region comprises at least one modification to extend the serum half-life. In some embodiments, the Fc region comprises a modification corresponding to the combination of substitutions M252Y / S254T / T256E (YTE) (e.g. as described in Dall’Acqua et al. J Immunol. (2002) 169:5171-5180). In some embodiments, the Fc region comprises a CH2-CH3 region comprising modification corresponding to the combination of substitutions M252Y / S254T / T256E.

[0099] In some embodiments, the Fc region comprises at least one modification to promote heterodimerization, reduce antibody-dependent cellular cytotoxicity, and / or extend the serum half-life.

[0100] In some embodiments the Fc region comprises a CH2-CH3 region comprising modification corresponding to the combination of substitutions L234A / L235A / P329G (LALAPG) and a ‘knob-into-hole’ modification. For example, in some embodiments the ‘knob’ Fc comprises the combination of substitutions L234A, L235A, P329G, S354C, and T366W, and the ‘hole’ Fc comprises the combination of substitutions L234A, L235A, P329G, Y349C, T366S, L368A and Y407V. The amino acid sequence of a CH2-CH3 region comprising LALAPG modifications and a knob mutation is set out in SEQ ID NO: 3 and 74. The amino acid sequence of a CH2-CH3 region comprising LALAPG modifications and a hole mutation is set out in SEQ ID NO: 4 and 75. In some embodiments, the fusion molecules of the present disclosure comprises an Fc region comprising or consisting of a polypeptide having an amino acid sequence according to SEQ ID NO: 3 or 74 and / or SEQ ID NO: 4 or 75. In some embodiments, the fusion molecules of the present disclosure comprises an Fc region comprising or consisting of a polypeptide having an amino acid sequence according to SEQ ID NO: 3 and / or SEQ ID NO: 4. In some embodiments, the fusion molecules of the present disclosure comprises an Fc region comprising or consisting of a polypeptide having an amino acid sequence according to SEQ ID NO: 74 and / or SEQ ID NO: 75.

[0101] In some embodiments, a fusion molecule of the present disclosure comprises a polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 3. In some embodiments, a fusion molecule of the present disclosure comprises a polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 74.

[0102] In some embodiments, a fusion molecule of the present disclosure comprises a polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 4. In some embodiments, a fusion molecule of the present disclosure comprises a polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 75.

[0103] In some embodiments, a fusion molecule of the present disclosure comprises an Fc-region comprising (i) a first polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 3, and (ii) a second polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 4.

[0104] In some embodiments, a fusion molecule of the present disclosure comprises an Fc-region comprising (i) a first polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 74, and (ii) a second polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 75. In some embodiments, the fusion molecule is a polypeptide complex comprising: (i) a first polypeptide comprising or consisting of a tumor antigen-binding moiety, an immune cell-binding moiety, and a first CH2-CH3 region, and (ii) a second polypeptide comprising or consisting of a second CH2-CH3 region and a cytokine. In some embodiments, said first and second CH2-CH3 regions are encoded by amino acid sequences according to SEQ ID NO: 3 and SEQ ID NO: 4. In some embodiments, the fusion molecule is a polypeptide complex comprising: (i) a first polypeptide comprising or consisting of a tumor antigen-binding moiety, an immune cell-binding moiety, and a first CH2-CH3 region, and (ii) a second polypeptide comprising or consisting of a second CH2-CH3 region and a cytokine. In some embodiments, said first and second CH2-CH3 regions are encoded by amino acid sequences according to SEQ ID NO: 74 and SEQ ID NO: 75.

[0105] In some embodiments the Fc region comprises a CH2-CH3 region comprising modification corresponding to the combination of substitutions L234A / L235A / P329G (LALAPG), M252Y / S254T / T256E (YTE) and a ‘knob-into-hole’ modification. For example, in some embodiments the ‘knob’ Fc comprises the combination of substitutions L234A, L235A, P329G, M252Y, S254T, T256E, S354C, and T366W, and the ‘hole’ Fc comprises the combination of substitutions L234A, L235A, P329G, M252Y, S254T, T256E, Y349C, T366S, L368A and Y407V.

[0106] The amino acid sequence of a CH2-CH3 region comprising LALAPG modifications, YTE modifications and a knob mutation is set out in SEQ ID NO: 5. The amino acid sequence of an CH2-CH3 region comprising LALAPG modifications, YTE modifications and a hole mutation is set out in SEQ ID NO: 6. In some embodiments, the fusion molecules of the present disclosure comprises an Fc region comprising or consisting of a polypeptide having an amino acid sequence according to SEQ ID NO: 5 and / or SEQ ID NO: 6.

[0107] In some embodiments, a fusion molecule of the present disclosure comprises a polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 5.

[0108] In some embodiments, a fusion molecule of the present disclosure comprises a polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 6.

[0109] In some embodiments, a fusion molecule of the present disclosure comprises an Fc-region comprising (i) a first polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 5, and (ii) a second polypeptide having an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the fusion molecule is a polypeptide complex comprising: (i) a first polypeptide comprising or consisting of a tumor antigen-binding moiety, an immune cell-binding moiety (e.g. a CD3- binding moiety), and a first CH2-CH3 region, and (ii) a second polypeptide comprising or consisting of a second CH2-CH3 region and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinbelow). In some embodiments, said first and second CH2-CH3 regions are encoded by amino acid sequences according to SEQ ID NO: 5 and SEQ ID NO: 6.

[0110] Cytokines

[0111] The fusion molecules of the present disclosure comprise a cytokine. Cytokines are small secreted signaling proteins. The term ‘cytokines’ include lymphokines (cytokines made by lymphocytes), monokines (cytokines made by monocytes), chemokines (cytokines with chemotactic activities), and interleukins (cytokines made by one leukocyte and acting on other leukocytes). The addition of a cytokine to fusion molecules of the present disclosure aims to support long-term immune cell survival, proliferation, and activation.

[0112] In some embodiments, the cytokine is a cytokine in the common gamma chain family of cytokines, which includes IL-2, IL-4, IL-7, IL-9, IL-15, IL-10 and IL-21. The cytokine-receptor complexes of this family share a cytokine receptor polypeptide; common cytokine receptor gamma chain (common gamma chain, yc, or CD132). In some embodiments, the cytokine is IL-15, IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-18 or IL- 21. Accordingly, disclosed herein is a fusion molecule comprising an immune cell-binding moiety, a tumor antigen-binding moiety, an Fc region and a cytokine, wherein the cytokine is IL-15, IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-18 or IL-21. In some embodiments, the cytokine is IL-15, IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-18, IL-21 , or an engineered version thereof.

[0113] In some embodiments, the cytokine is interleukin-15 (IL-15). The amino acid sequence of human IL-15 is set out in SEQ ID NO: 7. IL-15 is a 14-15 kDa glycoprotein essential for the development and maintenance of NK cells and memory CD8+T cells. In addition, IL-15 plays a critical role in the activation, effector functions, tissue residency, and senescence of CD8+T cells and has been found to activate NK cells. IL-15 structure and function is reviewed in Cai et al. (2023) (Front Pharmacol. 12; 14:1184703), which is hereby incorporated by reference in its entirety. In some embodiments, a fusion molecule as described herein may comprise a cytokine that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 7.

[0114] In some embodiments, the cytokine is interleukin-2 (IL-2). The amino acid sequence of human IL-2 is set out in SEQ ID NO: 8. IL-2 is secreted by CD4+ T cells and activated CD8+ T cells. Treatment with IL-2 is an approved immunotherapy for the treatment of cancer, and works by promoting proliferation and activity of effector immune cells such as T cells and NK cells (see e.g. Skrombolas and Frelinger, Expert Rev Clin Immunol. 2014; 10(2): 207-217, which is hereby incorporated by reference in its entirety). In some embodiments, a fusion molecule as described herein may comprise a cytokine that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 8.

[0115] In some embodiments, the cytokine is interleukin-4 (IL-4). The amino acid sequence of human IL-4 is set out in SEQ ID NO: 9. IL-4 is produced by multiple cellular sources (including activated CD4+T cells) and induces differentiation of naive T cells to helper T cells (Th2 cells). IL-4 also targets a broad range of target cells, including macrophages, hematopoietic precursor cells, stromal cells, NK cells, and fibroblasts, and can act as a potent anti-apoptotic factor (see e.g. Keegan. Fac. Rev. (2021) 7;10:71 , which is hereby incorporated by reference in its entirety). In some embodiments, a fusion molecule as described herein may comprise a cytokine that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 9.

[0116] In some embodiments, the cytokine is interleukin-7 (IL-7). The amino acid sequence of human IL-7 is set out in SEQ ID NO: 10. IL-7 is involved in multiple stages of immune cell development and maintenance. IL-7 biology is reviewed e.g. in Chen et al. Front Immunol. (2021) 12:747324, which is hereby incorporated by reference in its entirety. In some embodiments, a fusion molecule as described herein may comprise a cytokine that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 10.

[0117] In some embodiments, the cytokine is interleukin-9 (IL-9). The amino acid sequence of human IL-9 is set out in SEQ ID NO: 11 . IL-9 is involved in multiple stages of immune cell development and maintenance. IL-9 stimulates cell proliferation and supports the growth of multiple cell types including mast cells and T cells (see e.g. Chakraborty et al. Int J Mol Sci. (2019) 29;20(9):2113, which is hereby incorporated by reference in its entirety). In some embodiments, a fusion molecule as described herein may comprise a cytokine that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 11 .

[0118] In some embodiments, the cytokine is interleukin-12 (IL-12). The amino acid sequence of human IL-12 alpha subunits and IL-12 beta subunits is set out in SEQ ID NO: 12 and SEQ ID NO: 13, respectively. IL- 12 is involved in stimulation of both T cells and NK cells. Function of IL-12 and other members of the IL- 12 family is reviewed in Ullrich et al. EXCLI J. (2020) 19:1563-1589, which is hereby incorporated by reference in its entirety. In some embodiments, a fusion molecule as described herein may comprise a cytokine that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 12 and SEQ ID NO: 13.

[0119] In some embodiments, the cytokine is interleukin-18 (IL-18). The amino acid sequence of human IL-18 is set out in SEQ ID NO: 14. IL-18 is a costimulatory cytokine that stimulates the innate and acquired immune response, and is required for interferon y (IFN-y) production (see e.g. Ihim et al. Front Immunol. (2022) 11 ;13:919973, which is hereby incorporated by reference in its entirety). In some embodiments, a fusion molecule as described herein may comprise a cytokine that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 14.

[0120] In some embodiments, the cytokine is interleukin-21 (IL-21). The amino acid sequence of human IL-21 is set out in SEQ ID NO: 15. IL-21 induces cell division / proliferation of immune cells including NK cells and cytotoxic T cells. Several preclinical studies showed that IL-21 has antitumor activity via activation of NK and T or B cell responses (see e.g. Croce at al. J Immunol Res. (2015) 2015:696578, which is incorporated by reference in its entirety). In some embodiments, a fusion molecule as described herein may comprise a cytokine that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 15.

[0121] In some embodiments, the cytokine is interleukin-10 (IL-10). The amino acid sequence of human IL-10 is set out in SEQ ID NO:96. IL-10 is a major immune regulatory cytokine that acts on many cells of the immune system where it has profound anti-inflammatory functions, limiting excessive tissue disruption caused by inflammation. In some embodiments, a fusion molecule as described herein may comprise a cytokine that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 96.

[0122] In some embodiments, the cytokine is selected from the list consisting of: IL-15, IL-7, IL-18 and IL-21.

[0123] In some embodiments, the cytokine is an engineered cytokine. Engineered cytokines (e.g. sushi-IL-15, IL- 15-sushi or Neo-2 / 15) may have advantageous characteristics such as stronger immune cell activation ability or a longer half-life. Engineered cytokines are cytokines that have been modified ( / .e. contain sequence modifications, sequence substitutions, and / or fusion to other polypeptides) in order to improve certain characteristics. In some embodiments, the cytokine is an engineered version of a cytokine as described herein (e.g. an engineered version of IL-2, IL-4, IL-7, IL-9, IL-15, IL-10 and IL-21). In some embodiments, the cytokine is an engineered version of IL-15 or IL-7.

[0124] In some embodiments, the cytokine is an engineered version of IL-7. In some embodiments, the engineered version of IL-7 is referred to as Neo7.

[0125] In some embodiments, the cytokine is an IL-15 / sushi domain fusion construct, which may be referred to herein as "sushi-IL-15” or “IL-15-sushi”. IL-15 signals via binding to the IL-15 receptor (IL-15R), which comprises alpha, beta and gamma chains. IL-15Ralpha (e.g. human IL-15Ralpha, as set out in SEQ ID NO:45) binds with high affinity to IL-15. The sushi domain (also known as the complement control protein domain (CCP), e.g. as set out in SEQ ID NO:46) is a conserved domain found in IL-15Ralpha that is responsible for high affinity binding to IL-15. IL-15 / sushi domain fusion constructs have been developed to improve the potential of IL-15 for therapeutic use. Sushi-IL-15 and IL-15-sushi are IL-15 super-agonists with stronger immune cell activation ability compared to parental IL-15. For example, see Hu et al., Sci Rep. (2018) 16;8(1):7675. Doi: 10.1038 / s41598-018-25987-4, which is incorporated by reference in its entirety. Sushi-IL-15 (or IL-15-Sushi) as used herein refers to a fusion construct comprising a sushi domain (or fragment or variant thereof) and IL-15 (or a fragment or variant thereof). In some embodiments, sushi-IL-15 comprises the sushi domain from IL-15Ralpha (e.g. a sushi domain having the sequence of SEQ ID NO:46), and IL-15 (e.g. human IL-15, e.g. IL-15 having the sequence shown in SEQ ID NO:7). In some embodiments, the sushi domain and IL-15 are located on a single polypeptide chain. In some embodiments, the sushi domain and IL-15 are located on separate polypeptide chains. In embodiments where the sushi domain and IL-15 are located on separate polypeptide chains, the sushi domain and IL-15 will associate via non-covalent interactions (e.g. hydrogen bonds, ionic interactions). Sushi-IL-15 may be a polypeptide having the amino acid sequence set out in any one of SEQ ID NOs: 16, 17 and, 18. IL-15-sushi may be a polypeptide having the amino acid sequence set out in SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, a fusion molecule as described herein comprises a cytokine that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to any one of SEQ ID NOs: 16, 17, 18, 19, 20, or 92.

[0126] In some embodiments, the cytokine is Neo-2 / 15. Neo-2 / 15 reproduces the immunostimulatory function of IL-2 and IL-15 but is associated with reduced toxicity when compared with natural IL-2, by avoiding undesired preferential activation of CD25+immune cells. The amino acid sequence of Neo-2 / 15 is set out in SEQ ID NO: 21. Development of Neo-2 / 15 is described in Silva et al., Nature. (2019) 565(7738):186- 191 , which is hereby incorporated by reference in its entirety. In some embodiments, a fusion molecule as described herein may comprise a cytokine that comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 21.

[0127] Design of Fusion Molecules

[0128] In some embodiments, the components of the fusion molecule are directly connected. ‘Directly connected’ as used herein means that the amino acids encoding the individual components ( / .e. the immune cell-binding moiety, a tumor antigen-binding moiety, Fc region and / or cytokine) are adjacent to each other. By way of example, a polypeptide as described herein may comprise an amino acid sequence encoding the tumor antigen-binding moiety or immune cell-binding moiety adjacent to an amino acid sequence encoding the Fc region (e.g. an amino acid sequence encoding a CH2-CH3 region). In such embodiments, a fusion molecule has a peptide bond between the last amino acid of the tumor antigen-binding moiety or immune cell-binding moiety and the first amino acid of the Fc region.

[0129] The terms ‘first’ and ‘last’ amino acids as used herein depends on the orientation of the tumor antigenbinding moiety or immune cell-binding moiety and CH2-CH3 region. It will be appreciated that when the C-terminus of either the tumor antigen-binding moiety or immune cell-binding moiety is directly connected to the N-terminus of the CH2-CH3 region, a fusion molecule comprising a tumor antigen-binding moiety or immune cell-binding moiety directly fused to a CH2-CH3 region has a peptide bond between the last ( / .e. C-terminal) amino acid of the tumor antigen-binding moiety or immune cell-binding moiety and the first ( / .e. N-terminal) amino acid of the CH2-CH3 region.

[0130] In the fusion molecules described herein, the tumor antigen-binding moiety and / or the immune cellbinding moiety may be connected to the Fc region (e.g. connected directly or via a linker as described herein). In some embodiments the tumor antigen-binding moiety is connected to the Fc region ( / .e. the tumor antigen-binding moiety and a CH2-CH3 domain form a single polypeptide with multiple domains). In some embodiments the immune cell-binding moiety is connected to the Fc region ( / .e. the immune cellbinding moiety and a CH2-CH3 domain form a single polypeptide with multiple domains).

[0131] “Y-Format” Fusion Molecules

[0132] In some embodiments, a fusion molecule is a polypeptide complex comprising a first polypeptide comprising a tumor antigen-binding moiety and a CH2-CH3 domain, and a second polypeptide comprising an immune cell-binding moiety and a CH2-CH3 domain. In some embodiments, the first and / or second polypeptide further comprises a cytokine, optionally an engineered cytokine.

[0133] In some embodiments, a fusion molecule is a polypeptide complex comprising a first polypeptide comprising a tumor antigen-binding moiety and a CH2-CH3 domain, and a second polypeptide comprising an immune cell-binding moiety, a CH2-CH3 domain and a cytokine, optionally an engineered cytokine.

[0134] In some embodiments, a fusion molecule is a polypeptide complex comprising a first polypeptide comprising an immune cell-binding moiety and a CH2-CH3 domain, and a second polypeptide comprising a tumor antigen-binding moiety, a CH2-CH3 domain and a cytokine, optionally an engineered cytokine. In some embodiments, the tumor antigen-binding moiety is a scFv and the immune cell-binding moiety is an scFv. In some embodiments, the tumor antigen-binding moiety is a VHH and the immune cell-binding moiety is a scFv. In some embodiments the tumor antigen-binding moiety is a Fab and the immune cellbinding moiety is a scFv.

[0135] In some embodiments, the fusion molecule comprises a linker sequence (e.g. as described herein) between the CH2-CH3 domain and the cytokine, and / or between the tumor antigen-binding moiety and the CH2-CH3 region, and / or the immune cell-binding moiety and the CH2-CH3 region, and / or C-terminal to a CH2-CH3 region. The linker may be any linker described herein, for example, a linker comprising or consisting of the amino acid sequence of SEQ ID NO:22, 23, 24, 25, 26, 27, 28, 90, 68 to 73 or 91. In some embodiments, the fusion molecule comprises a linker sequence between the CH2-CH3 region and the cytokine, wherein the linker sequence comprises or consists of the amino acid sequence of SEQ ID NO:24.

[0136] In some embodiments, the fusion molecule comprises a linker sequence between the CH2-CH3 domain and the cytokine, wherein the linker comprises or consists of the amino acid sequence of any one of SEQ ID NO:68 to 73 or 91.

[0137] In some embodiments, the fusion molecule is a polypeptide complex comprising, or consisting of: (i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety (e.g. a DLL3, EpCAM, TROP2, SEZ6, KLK2, PSMA,STEAP1 , STEAP2, CLDN6 and MUC16 binding moiety as described herein), a first CH2-CH3 region, and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove e.g. comprising, or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 7, 16, 17, 18, 19 or 20 as described hereinabove); and

[0138] (ii) a second polypeptide comprising from N to C terminus: an immune cell-binding moiety (e.g. a CD2, CD3, CD16, CD28, CD54, CD137 or CD335 binding moiety as described herein), and a second CH2-CH3 region.

[0139] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 35.

[0140] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 36.

[0141] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 37.

[0142] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 38.

[0143] In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising, or consisting of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 36, and a second polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 35.

[0144] In some embodiments, a fusion molecule as described herein comprises, or consists of, a first polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 37, and a second polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 35.

[0145] In some embodiments, a fusion molecule as described herein comprises, or consists of, a first polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 38, and a second polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 35.

[0146] “P-Format” and “K-Format” Fusion Molecules

[0147] In some embodiments, a fusion molecule comprises a polypeptide comprising a tumor antigen-binding moiety, an immune cell-binding moiety and a CH2-CH3 domain. In some embodiments, a fusion molecule comprises a first polypeptide comprising a tumor antigen-binding moiety, an immune cell-binding moiety and a CH2-CH3 domain and a second polypeptide comprising a CH2-CH3 domain. In some embodiments, the first and / or second polypeptide further comprises a cytokine, optionally an engineered cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove, e.g. comprising, or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 7, 16, 17, 18, 19 or 20 as described hereinabove).

[0148] In some embodiments, a fusion molecule comprises a polypeptide comprising a cytokine and a CH2-CH3 domain.

[0149] In some preferred embodiments, the Fc region is a polypeptide complex comprising two constituent CH2- CH3 region polypeptides, and the tumor antigen-binding domain and the cytokine are each connected to a CH2-CH3 region on different constituent polypeptides of the Fc region. In other words, in some embodiments the fusion molecules of the present disclosure comprise a polypeptide comprising an Fc region (e.g. a CH2-CH3 domain) and a cytokine, wherein said polypeptide does not comprise a tumor antigen-binding domain. Accordingly, in such embodiments it will be appreciated that the fusion molecules also comprise a polypeptide comprising an Fc region (e.g. a CH2-CH3 domain) and a tumor antigen-binding domain, wherein said polypeptide does not comprise a cytokine.

[0150] In some embodiments, the fusion molecule comprises a linker sequence (e.g. as described herein) between the CH2-CH3 domain and the cytokine and / or between the tumor antigen-binding moiety and the CH2-CH3 region, and / or the immune cell-binding moiety and the CH2-CH3 region, and / or between the tumor antigen-binding moiety and the immune cell-binding moiety, and / or C-terminal to a CH2-CH3 region. The linker may be any linker described herein, for example, a linker comprising or consisting of the amino acid sequence of SEQ ID NO:22, 23, 24, 25, 26, 27, 28, 90, 68 to 73 or 91 . In some embodiments, the fusion molecule comprises a linker sequence between the CH2-CH3 region and the cytokine, wherein the linker sequence comprises or consists of the amino acid sequence of SEQ ID NO:24. In some embodiments, the fusion molecule comprises a linker sequence between the CH2-CH3 region and the cytokine, wherein the linker sequence comprises or consists of the amino acid sequence of SEQ ID NO:25.

[0151] In some embodiments, the fusion molecule comprises a linker sequence between the CH2-CH3 domain and the cytokine, wherein the linker comprises or consists of the amino acid sequence of any one of SEQ ID NO:68 to 73 or 91 . In some embodiments, the fusion molecule comprises a linker sequence between the CH2-CH3 region and the cytokine, wherein the linker sequence comprises or consists of the amino acid sequence of SEQ ID NO:91 .

[0152] In some preferred embodiments, the fusion molecule is a polypeptide complex comprising, or consisting of:

[0153] (i) a first polypeptide comprising a tumor antigen-binding moiety, an immune cell-binding moiety (e.g. a CD3-binding moiety), and a first CH2-CH3 region, and

[0154] (ii) a second polypeptide comprising a second CH2-CH3 region and a cytokine (e.g. IL-15, Sushi- IL-15 or IL-15-Sushi as described hereinabove, e.g. comprising, or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 7, 16, 17, 18, 19 or 20 as described hereinabove).

[0155] In some such embodiments, the first and / or the second CH2-CH3 region comprise at least one modification to promote heterodimerization, prevent antibody-dependent cellular cytotoxicity and / or extend serum half-life (e.g. the LALAPG, YTE and ‘knob-into-hole’ modifications described hereinabove). In some embodiments, the first and / or the second CH2-CH3 region comprise knob-into-hole modifications.

[0156] In some preferred embodiments the tumor antigen-binding moiety and / or the immune cell-binding moiety are connected to the N-terminus of the Fc region. In some embodiments the tumor antigen-binding moiety and / or the immune cell-binding moiety are connected to the N-terminus of a CH2 region.

[0157] “P-format” fusion molecules

[0158] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, an immune cell-binding moiety, and a CH2-CH3 region.

[0159] In some embodiments the cytokine is connected to the N-terminus of the Fc region. In some embodiments, the cytokine is connected to the N-terminus of a CH2 region.

[0160] In some preferred embodiments the cytokine is connected to the C-terminus of the Fc region. Accordingly, in some preferred embodiments, the cytokine is connected to the C-terminus of a CH3 region. It will be appreciated that a linker (e.g. a linker as described herein) may be present between any of the regions or moieties of the fusion molecule.

[0161] In some embodiments, the fusion molecule comprises a linker sequence between the CH2-CH3 domain and the cytokine, and / or between the tumor antigen-binding moiety and the CH2-CH3 region, and / or the immune cell-binding moiety and the CH2-CH3 region, and / or between the tumor antigen-binding moiety and the immune cell-binding moiety, and / or C-terminal to a CH2-CH3 region, and / or N-terminal to a CH2- CH3 region. The linker may be any linker described herein, for example, a linker comprising or consisting of the amino acid sequence of SEQ ID NO:22, 23, 24, 25, 26, 27, 28 or 90. The linker may be any linker described herein, for example, a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 68 to 73 or 91 . In some embodiments, the fusion molecule comprises a linker sequence between the CH2-CH3 region and the cytokine, wherein the linker sequence comprises or consists of the amino acid sequence of SEQ ID NO:24 or 91 .

[0162] In some embodiments, the fusion molecule is a polypeptide complex comprising, or consisting of:

[0163] (i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, an immune cell-binding moiety (e.g. a CD3-binding moiety), and a first CH2-CH3 region, and

[0164] (ii) a second polypeptide comprising from N to C terminus: a second CH2-CH3 region and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove, e.g. comprising, or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 7, 16, 17, 18, 19, 20 or 92 as described hereinabove).

[0165] In some such embodiments, the fusion molecule is a polypeptide complex comprising, or consisting of:

[0166] (i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, an immune cell-binding moiety (e.g. a CD3-binding moiety), and a CH2-CH3 region comprising a ‘hole’ modification, and

[0167] (ii) a second polypeptide comprising from N to C terminus: a CH2-CH3 region comprising a ‘knob’ modification and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove, e.g. comprising, or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 7, 16, 17, 18, 19, 20 or 92 as described hereinabove).

[0168] The position of the tumor antigen-binding moiety and the immune cell-binding moiety may be swapped. Accordingly, in some embodiments, the fusion molecule is a polypeptide complex comprising, or consisting of:

[0169] (i) a first polypeptide comprising from N to C terminus: an immune cell-binding moiety (e.g. a CD3-binding moiety), a tumor antigen-binding moiety, and a first CH2-CH3 region (e.g. a CH2-CH3 region comprising a ‘hole’ modification), and

[0170] (ii) a second polypeptide comprising from N to C terminus: a second CH2-CH3 region (e.g. a CH2-CH3 region comprising a ‘knob’ modification) and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove, e.g. comprising, or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 7, 16, 17, 18, 19, 20 or 92 as described hereinabove). In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising, from N-terminus to C-terminus: an tumor-antigen binding moiety, an immune cell-binding moiety and a CH2- CH3 region. In some embodiments, the tumor antigen binding moiety is a scFv. In some embodiments, the tumor antigen binding moiety is a DLL3 binding moiety. In some embodiments, the tumor antigenbinding moiety comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the immune cellbinding moiety is an scFv. In some embodiments, the immune cell-binding moiety is a CD3 scFv. In some embodiments, the immune cell-binding moiety comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 1. In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising an CH2-CH3 region. In some embodiments, the CH2-CH3 region comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to 4 or 75.

[0171] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising an immune cell binding moiety. In some embodiments, the immune cell binding moiety comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 81 .

[0172] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising, or consisting of, a CH2-CH3 region. In some embodiments, a fusion molecule as described herein comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 3 or 74.

[0173] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising from N to C terminus: an anti-DLL3 scFv, an anti-CD3 scFv, and a CH2-CH3 region. In some embodiments, a fusion molecule as described herein comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 39

[0174] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising from N to C terminus: a CH2-CH3 region and a cytokine.

[0175] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising from N to C terminus: a CH2-CH3 region and Sushi-IL-15. In some embodiments, the CH2-CH3 domain comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 3 or 74. In some embodiments, the sushi-IL15 comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 16 or 92. In some embodiments, a fusion molecule as described herein comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 80. In some embodiments, a fusion molecule as described herein comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 95.

[0176] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising from N to C terminus: a CH2-CH3 region and IL-15. In some embodiments, a fusion molecule as described herein comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 40.

[0177] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising from N to C terminus: a CH2-CH3 region and Sushi-IL-15, wherein the Sushi-IL-15 comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 16, 17, 18 or 92.

[0178] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising from N to C terminus: a CH2-CH3 region and Sushi-IL-15. In some embodiments, a fusion molecule as described herein comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 41 .

[0179] In some embodiments, a fusion molecule as described herein comprises a polypeptide comprising from N to C terminus: a CH2-CH3 region and IL-15-Sushi, wherein the IL-15-Sushi comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 19 or 20.

[0180] In some embodiments, a fusion molecule as described herein comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 39, and an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 40. In some embodiments, a fusion molecule as described herein comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 39, and an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 41 .

[0181] In some embodiments, a fusion molecule as described herein comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 80, and an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 81 .

[0182] In some embodiments, a fusion molecule as described herein comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 94, and an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 95.

[0183] “K-format” fusion molecules

[0184] In some embodiments the tumor antigen-binding moiety and / or the immune cell-binding moiety are connected to the C-terminus of the Fc region. In some embodiments the tumor antigen-binding moiety and / or the immune cell-binding moiety are connected to the C-terminus of a CH3 region. By way of example, in some embodiments, a CD3-binding moiety is connected to the C-terminus of a CH3 region. In some embodiments, the tumor antigen-binding moiety is connected to the N-terminus of the Fc region and the immune cell-binding moiety is connected to the C-terminus of the Fc region. In some embodiments, the tumor antigen-binding moiety is connected to the N-terminus of a CH2 region, and the immune cell-binding moiety is connected to the C-terminus of a CH3 region. In some embodiments, the immune cell-binding moiety is connected to the N-terminus of the Fc region and the tumor antigen-binding moiety is connected to the C-terminus of the Fc region. In some embodiments, the immune cell-binding moiety is connected to the N-terminus of a CH2 region, and the tumor antigen-binding moiety is connected to the C-terminus of a CH3 region.

[0185] In some embodiments, the fusion molecule is a polypeptide complex comprising, or consisting of:

[0186] (i) a first polypeptide comprising a tumor antigen-binding moiety, a first CH2-CH3 region, and an immune cell-binding moiety,

[0187] (ii) a second polypeptide comprising a second CH2-CH3 region and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove).

[0188] In some embodiments, the fusion molecule is a polypeptide complex comprising, or consisting of: (i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, a first CH2-CH3 region, and an immune cell-binding moiety,

[0189] (ii) a second polypeptide comprising from N to C terminus: a second CH2-CH3 region and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove).

[0190] It will be appreciated that the first and second polypeptide chains associate together to form a single polypeptide complex. For example, via association between the CH2-CH3 region of each polypeptide chain.

[0191] It will be appreciated that a linker (e.g. a linker as described herein) may be present between any of the regions or moieties of the fusion molecule.

[0192] For example, in some embodiments, the fusion molecule is a polypeptide complex comprising, or consisting of:

[0193] (i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, a linker (e.g. as described herein), a first CH2-CH3 region, a linker, and an immune cell-binding moiety,

[0194] (ii) a second polypeptide comprising from N to C terminus a second CH2-CH3 region, a linker, and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove).

[0195] For example, in some embodiments, the fusion molecule is a polypeptide complex comprising, or consisting of:

[0196] (i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, a first CH2-CH3 region, a linker, and an immune cell-binding moiety,

[0197] (ii) a second polypeptide comprising from N to C terminus a second CH2-CH3 region, a linker, and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove).

[0198] In some embodiments, the fusion molecule comprises a linker sequence between the CH2-CH3 domain and the cytokine, and / or between the tumor antigen-binding moiety and the CH2-CH3 region, and / or the immune cell-binding moiety and the CH2-CH3 region, and / or C-terminal to a CH2-CH3 region, and / or N- terminal to a CH2-CH3 region. The linker may be any linker described herein, for example, a linker comprising or consisting of the amino acid sequence of SEQ ID NO:22, 23, 24, 25, 26, 27, 28 or 90. The linker may be any linker described herein, for example, a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 68 to 73 or 91. In some embodiments, the fusion molecule comprises a linker sequence between the CH2-CH3 region and the cytokine, wherein the linker sequence comprises or consists of the amino acid sequence of SEQ ID NO:24 or 25.

[0199] In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, a CH2-CH3 region, and an immune cell-binding moiety. In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, a CH2-CH3 region, and a CD3-binding moiety. In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, a CH2-CH3 region, and an immune cell-binding moiety. In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, a CH2-CH3 region, and a CD3-binding moiety. In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N-terminus to C terminus: a DLL3-binding moiety, a CH2-CH3 region, and a CD3-binding moiety. In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N-terminus to C terminus: an anti-DLL3 VHH, a CH2-CH3 region, and an anti-CD3 scFv. In some embodiments, the DLL3-binding moiety comprises, or consists of, an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 47. In some embodiments, the CH2-CH3 region comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 3 or 74. In some embodiments, the CD3-binding moiety comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 1. In some embodiments, a fusion molecule as described herein comprises, or consists of, a first polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 76 or 77. In some embodiments, a fusion molecule as described herein comprises, or consists of, a first polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 43.

[0200] In some embodiments, a fusion molecule as described herein comprises a second polypeptide comprising from N to C terminus: a CH2-CH3 region, and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15- Sushi as described hereinabove). In some embodiments, a fusion molecule as described herein comprises a second polypeptide comprising from N to C terminus: a CH2-CH3 region, and a sushi-IL-15. In some embodiments, the CH2-CH3 region comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 4 or 74. In some embodiments, the sushi-IL-15 comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 16 or 92. In some embodiments, a fusion molecule as described herein comprises, or consists of, a first polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 77 or 79.

[0201] In some embodiments, a fusion molecule as described herein comprises a second polypeptide comprising from N to C terminus: a CH2-CH3 region, and a cytokine e.g. IL-15, Sushi-IL-15 or IL-15- Sushi as described hereinabove). In some embodiments, a fusion molecule as described herein comprises a second polypeptide comprising from N to C terminus: a CH2-CH3 region, and a IL-15. In some embodiments, the CH2-CH3 region comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 4 or 74. In some embodiments, the sushi-IL-15 comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 7. In some embodiments, a fusion molecule as described herein comprises, or consists of, a first polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 40.

[0202] In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, a CH2-CH3 region, and an immune cell-binding moiety, and a second polypeptide comprising from N to C terminus a CH2-CH3 region, and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove). In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N to C terminus a tumor antigen-binding moiety, a CH2-CH3 region, and a CD3-binding moiety, and a second polypeptide comprising from N to C terminus a CH2-CH3 region, and a cytokine e.g. IL-15, Sushi-IL-15 or IL-15- Sushi as described hereinabove).

[0203] In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N-terminus to C terminus: a DLL3-binding moiety, a CH2-CH3 region, and a CD3-binding moiety and a second polypeptide comprising from N to C terminus a CH2-CH3 region, and a sushi-IL-15. In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N-terminus to C terminus: an anti-DLL3 VHH, a CH2-CH3 region, and an anti-CD3 scFv and a second polypeptide comprising from N to C terminus a CH2-CH3 region, and a sushi-IL-15. In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N- terminus to C terminus: an anti-DLL3 VHH, optionally a first linker, a CH2-CH3 region, optionally a second linker, and an anti-CD3 scFv and a second polypeptide comprising from N to C terminus optionally a third linker, a CH2-CH3 region, optionally a fourth linker, and a sushi-IL-15. It will be appreciated that the fusion molecule may comprise any combination of the first to fourth linkers described above. The linker may be any linker described herein. In some embodiments, a fusion molecule as described herein comprises, or consists of, a first polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 76 and a second polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 77.

[0204] In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N-terminus to C terminus: a DLL3-binding moiety, a CH2-CH3 region, and a CD3-binding moiety and a second polypeptide comprising from N to C terminus a CH2-CH3 region, and a sushi-IL-15. In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N-terminus to C terminus an anti-DLL3 VHH, a CH2-CH3 region, and an anti-CD3 scFv and a second polypeptide comprising from N to C terminus a CH2-CH3 region, and a sushi-IL-15. In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N- terminus to C terminus: an anti-DLL3 VHH, optionally a first linker, a CH2-CH3 region, optionally a second linker, and an anti-CD3 scFv and a second polypeptide comprising from N to C terminus optionally a third linker, a CH2-CH3 region, optionally a fourth linker, and a sushi-IL-15. It will be appreciated that the fusion molecule may comprise any combination of the first to fourth linkers described above. The linker may be any linker described herein. In some embodiments, a fusion molecule as described herein comprises, or consists of, a first polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 78 and a second polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 79.

[0205] In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N-terminus to C terminus a DLL3-binding moiety, a CH2-CH3 region, and a CD3-binding moiety and a second polypeptide comprising from N to C terminus a CH2-CH3 region, and IL-15. In some embodiments, a fusion molecule as described herein comprises a first polypeptide comprising from N- terminus to C terminus an anti-DLL3 VHH, a CH2-CH3 region, and an anti-CD3 scFv and a second polypeptide comprising from N to C terminus a CH2-CH3 region, and a IL-15. In some embodiments, a fusion molecule as described herein comprises, or consists of, a first polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 43 and a second polypeptide comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 40.

[0206] In some embodiments, a fusion molecule as described herein comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 40, and an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 43.

[0207] In some embodiments, a fusion molecule as described herein comprises, or consists of an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 44, and an amino acid sequence having at least 70% amino acid sequence identity, e.g. one of >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to SEQ ID NO: 43.

[0208] Linkers

[0209] In some embodiments, the fusion molecules of the present disclosure comprise one or more linker sequences between sequences of amino acids encoding components of the fusion molecules. The term “linker” as used herein refers to a component (e.g. a short amino acid sequence) that connects components (e.g. immune cell-binding moiety, a tumor antigen-binding moiety, an Fc region, and / or a cytokine) of the fusion molecule. For example, the cytokine may be connected to the Fc region via a linker. The term “linker” as used herein may also encompass a component (e.g. a short amino acid sequence) which is connected to one component of the fusion molecule. For example, a linker may be connected to an Fc region.

[0210] In some embodiments, fusion molecules comprise a tumor antigen-binding moiety which is connected to the immune cell-binding moiety via a linker. In some embodiments, the tumor antigen-binding moiety (e.g. a scFv, VH domain, Fab or nanobody) is connected to the immune cell-binding moiety (e.g. a scFv, VH domain, Fab or nanobody) via a linker.

[0211] Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids. In some embodiments, the linker is a polypeptide linker having a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids. In some embodiments, the linker comprises or consists of an amino acid sequence according to any of SEQ ID NOs: 22-32, 68-73, 90 or 91 . Accordingly, in some embodiments, the tumor antigen-binding moiety is connected to the immune cell-binding moiety via a linker, wherein the linker comprises or consists of an amino acid sequence according to any of SEQ ID NOs: 22-32, 68-73, 90 or 91.

[0212] A linker moiety according to the present disclosure may be a non-cleavable moiety. Non-cleavable linkers remain inert in common chemical and enzymatic environments in the body. Non-cleavable linkers include linkers having an amino acid sequence lacking a protease cleavage site.

[0213] In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence.

[0214] Flexible linkers are known to the skilled person, and several are identified in Chen etal., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and / or serine residues. In some embodiments, the flexible linker comprises or consists of an amino acid sequence according to any of SEQ ID NOs: 22, 24 or 25.

[0215] In some embodiments, the flexible linker comprises, or consists of, an amino acid sequence according to SEQ ID NO: 22. Accordingly, in some embodiments, the tumor antigen-binding moiety is connected to the immune cell-binding moiety via a linker which comprises or consists of an amino acid sequence according to SEQ ID NO: 22. In some embodiments, the flexible linker comprises or consists of an amino acid sequence according to SEQ ID NO: 24 or 25.

[0216] In some embodiments, fusion molecules comprise a cytokine connected to an Fc region (e.g. a polypeptide comprising a CH2-CH3 region) via a linker. In some embodiments, the cytokine is connected to the Fc region via a linker which comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 29, 31 , 32, 68-73, 90 or 91 . In some such embodiments, the cytokine is connected to a CH2-CH3 region comprising a ‘knob’ mutation via a linker which comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 29, 31 ,32, 68-73, 90 or 91 . Accordingly, in some embodiments, a fusion molecule according to the present invention comprises a polypeptide comprising from N to C terminus a second CH2-CH3 region, a linker which comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 29, 31 , 32, 68-73, 90 or 91 , and a cytokine.

[0217] In some embodiments, the cytokine is connected to the Fc region via a flexible linker. In some such embodiments, the cytokine is connected to the Fc region via a linker which comprises or consists of an amino acid sequence according to SEQ ID NO: 22, 24 or 25. By way of example, IL-15 (or an engineered cytokine e.g. sushi-IL-15 or IL-15-sushi) may be connected to the Fc region via a linker which comprises or consists of an amino acid sequence according to SEQ ID NO: 22, 24 or 25.

[0218] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n or (GxS)nGm; wherein G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1 , 2 or 3. In some embodiments, the linker sequence comprises one or more (e.g. 1 , 2, 3, 4, 5 or 6) copies (e.g. in tandem) of the sequence motif G4S. In some embodiments, the linker sequence comprises or consists of (648)3 or (648)4.

[0219] In some embodiments, linkers may be used to increase the stability of the cytokine. In some such embodiments, fusion molecules are designed with a first and second linker, so that a disulfide bond forms between a first linker on a first CH2-CH3 region and a second linker on a second CH2-CH3 region. By way of example, in some embodiments a fusion molecule comprises a first linker which connects the cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove, e.g. comprising, or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 7, 16, 17, 18, 19, 20 or 92 as described hereinabove) to a first CH2-CH3 region comprising a ‘knob’ mutation (Fc-knob), and a second linker on a second CH2-CH3 region comprising a ‘hole’ mutation (Fc- hole), wherein a disulfide bond forms between said linkers. In some embodiments a fusion molecule comprises a first linker which connects the IL-15, sushi-IL-15 or IL-15-sushi to a Fc-knob, and a second linker on an Fc-hole.

[0220] In some embodiments, the cytokine is connected to the CH2-CH3 region via a linker comprising the amino acid sequence set out in SEQ ID NO: 27, 29 or 31 . In some embodiments a fusion molecule comprises a first linker comprising the amino acid sequence set out in SEQ ID NO: 28, and a second linker comprising the amino acid sequence set out in SEQ ID NO: 27. In some embodiments a fusion molecule comprises a first linker comprising the amino acid sequence set out in SEQ ID NO: 28 on a first CH2-CH3 region comprising a ‘hole’ mutation (Fc-hole), and a second linker comprising the amino acid sequence set out in SEQ ID NO: 27 which connects the cytokine to a second CH2-CH3 region comprising a ‘knob’ mutation (Fc-knob).

[0221] In some embodiments a fusion molecule comprises a first linker comprising the amino acid sequence set out in SEQ ID NO: 30, and a second linker comprising the amino acid sequence set out in SEQ ID NO: 29. In some embodiments a fusion molecule comprises a first linker comprising the amino acid sequence set out in SEQ ID NO: 30 on a first CH2-CH3 region comprising a ‘hole’ mutation (Fc-hole), and a second linker comprising the amino acid sequence set out in SEQ ID NO: 29 which connects the cytokine to a second CH2-CH3 region comprising a ‘knob’ mutation (Fc-knob).

[0222] In some embodiments a fusion molecule comprises a first linker comprising the amino acid sequence set out in SEQ ID NO: 32, and a second linker comprising the amino acid sequence set out in SEQ ID NO: 31 . In some embodiments a fusion molecule comprises and a first linker comprising the amino acid sequence set out in SEQ ID NO: 32 on a first CH2-CH3 region comprising a ‘hole’ mutation (Fc-hole), and a second linker comprising the amino acid sequence set out in SEQ ID NO: 31 which connects the cytokine to a second CH2-CH3 region comprising a ‘knob’ mutation (Fc-knob).

[0223] It will be appreciated that the embodiments described above, the CH2-CH3 regions comprising the knob and hole mutations may be used interchangeably. For example, in some embodiments a fusion molecule comprises a first linker on a first CH2-CH3 region comprising a ‘knob’ mutation, and comprises a second linker which connects the cytokine to a second CH2-CH3 region comprising a ‘hole’ mutation.

[0224] In some embodiments, the fusion molecule is a polypeptide complex comprising:

[0225] (i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, an immune cell-binding moiety, a first CH2-CH3 region, and a first linker (e.g. a linker comprising or consisting of the amino acid sequence set out in SEQ ID NO: 28, 30 or 32), and

[0226] (ii) a second polypeptide comprising from N to C terminus: a second CH2-CH3 region, a second linker (e.g. a linker comprising or consisting of the amino acid sequence set out in SEQ ID NO: 27, 29 or 31) and a cytokine.

[0227] By way of example, in some embodiments, the fusion molecule is a polypeptide complex comprising:

[0228] (i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, an immune cell-binding moiety, a CH2-CH3 region comprising a ‘hole’ modification, and a linker comprising or consisting of the amino acid sequence set out in SEQ ID NO: 28, and

[0229] (ii) a second polypeptide comprising from N to C terminus: a CH2-CH3 region comprising a ‘knob’ modification, a linker comprising or consisting of the amino acid sequence set out in SEQ ID NO: 27 and a cytokine. The fusion molecules of the present disclosure may additionally comprise further amino acids or sequences of amino acids. For example, the fusion molecules of the present disclosure may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule / polypeptide.

[0230] Functional properties

[0231] Fusion molecules described herein may be characterised by reference to certain functional properties. In some embodiments, a fusion molecule described herein may possess one or more of the following properties:

[0232] Binds to an immune cell

[0233] Binds to a T cell

[0234] Binds CD3

[0235] Binds to a NK cell

[0236] Activates T cells

[0237] Activates NK cells

[0238] Induces T-cell dependent cellular cytotoxicity

[0239] Binds to a cancer cell antigen

[0240] Binds to a cancer-associated antigen

[0241] Binds to DLL3

[0242] Binds to cytokine receptors

[0243] Binds to IL-2 receptor beta

[0244] Binds to IL-2 receptor gamma

[0245] Reduces tumor size

[0246] Induce memory T cells

[0247] Reduces T cell exhaustion

[0248] Less sensitive to T-cell exhaustion compared to standard of care

[0249] Ability to re-stimulate exhausted T cells

[0250] Enhance T cell activation (e.g. as compared to a control antibody / standard of care)

[0251] Increase T cell proliferation (e.g. as compared to a control antibody / standard of care)

[0252] Promote T cell fitness and persistence

[0253] Induce NK cells / T-cells proliferation

[0254] Promote long term survival of immune cells (e.g. as compared to a control antibody / standard of care) Sustain cytotoxic activity

[0255] Triggers long term T-cell proliferation (e.g. as compared to a control antibody / standard of care) Maintain long term T-cell viability (e.g. as compared to a control antibody / standard of care)

[0256] It will be appreciated that a given fusion molecule may display more than one of the properties recited in the preceding paragraph. A given fusion molecule may be evaluated for the properties recited in the preceding paragraph using suitable assays. For example, the assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays. In some embodiments, the assays may be e.g. in vivo assays, i.e. performed in non-human animals e.g. Mus musculus. In some embodiments, the assays may be e.g. ex vivo assays, i.e. performed using cells / tissue / an organ obtained from a subject. Such assays may be utilised to screen for fusion molecules with a desired functional property.

[0257] Where assays are cell-based assays, they may comprise treating cells with a given fusion molecule in order to determine whether the fusion molecule displays one or more of the recited properties. Assays may employ species labelled with detectable entities in order to facilitate their detection. Assays may comprise evaluating the recited properties following treatment of cells separately with a range of quantities / concentrations of a given fusion molecule (e.g. a dilution series). When the tumor antigenbinding moiety comprises an antigen-binding molecule, it will be appreciated that the cells preferably express the target antigen for the antigen-binding molecule.

[0258] Analysis of the results of such assays may comprise determining the concentration at which 50% of the maximal level of the relevant activity is attained. The concentration of a given agent at which 50% of the maximal level of the relevant activity is attained may be referred to as the ‘half-maximal effective concentration’ of the agent in relation to the relevant activity, which may also be referred to as the ‘EC50’. By way of illustration, the EC50 of a given fusion molecule for binding to DLL3 may be the concentration of the fusion molecule at which 50% of the maximal level of binding to DLL3 is achieved.

[0259] Where the functional properties of agents are compared (e.g. where the fusion molecules of the present disclosure are compared with other polypeptides, such as their constituent polypeptides), comparisons are performed at equivalent concentrations and / or quantity of the relevant agents.

[0260] The ability of a given polypeptide to bind specifically to a given molecule can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol. (2012) 907:41 1-442), Bio-Layer Interferometry (BLI; see e.g. Lad et al., J Biomol Screen (2015) 20(4):498-507), flow cytometry, radiolabeled antigen-binding assay (RIA), or by an enzyme-linked immunosorbent assay. Through such analysis binding to a given molecule can be measured and quantified. In some embodiments, the binding may be the response detected in a given assay.

[0261] Binding affinity of an antigen-binding molecule / moiety for its target is often described in terms of its dissociation constant (KD). Binding affinity can be measured by methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442; or Rich et al., Anal Biochem. (2008) 373(1):112-20), Bio-Layer Interferometry (see e.g. Lad et al., J Biomol Screen (2015) 20(4):498-507; or Concepcion et al., Comb Chem High Throughput Screen. (2009) 12(8)791-800), MicroScale Thermophoresis (MST) analysis (see e.g. Jerabek-Willemsen et al., Assay Drug Dev Technol. (2011) 9(4):342-353), or by a radiolabelled antigen-binding assay (RIA).

[0262] The ability of a given antigen-binding molecule to impact T-cell exhaustion (e.g. to reduce T cell exhaustion, re-stimulate exhausted T cells) can be measured using known methods, e.g. the methods described in Example 3.3. or 3.4. herein. In some embodiments, the antigen-binding molecules of the present invention are less sensitive to T-cell exhaustion compared to the standard of care, wherein the standard of care may be an equivalent antigen-binding molecule not comprising a cytokine (e.g. tarlatamab). In some embodiments, the antigen-binding molecules of the present invention demonstrate improved ability to re-stimulate exhausted T cells compared to equivalent antigen-binding molecules not comprising a cytokine, where standard of care may be an equivalent antibody without a cytokine (e.g. tarlatamab).

[0263] Therapeutic / prophylactic applications

[0264] The fusion molecules, polynucleotides, vectors, cells and compositions described herein find use in therapeutic and prophylactic intervention for disease, e.g. therapeutic and prophylactic intervention for cancer.

[0265] The present disclosure provides a fusion molecule or composition described herein for use in a method of medical treatment or prophylaxis. In some embodiments is a method of treating or preventing a disease or condition, comprising administering a fusion molecule comprising an immune cell engager and a cytokine as described herein, or a polynucleotide, vector, cell or pharmaceutical composition as described herein, to a patient in need thereof. In some embodiments is a fusion molecule comprising an immune cell engager and a cytokine as described herein, or a polynucleotides, vector, cell or pharmaceutical composition as described herein, for use in a method of treating or preventing a disease or condition. In some embodiments is the use of a fusion molecule comprising an immune cell engager and a cytokine as described herein, or a polynucleotide, vector, cell or pharmaceutical composition as described herein, in the manufacture of a medicament for treating or preventing a disease or condition.

[0266] Also provided is a fusion molecule or composition described herein for use in a method of treating or preventing a cancer (e.g. a cancer described herein). Also provided is the use of a fusion molecule or composition described herein in the manufacture of a medicament for treating or preventing a cancer (e.g. a cancer described herein). Also provided is a method of treating or preventing a cancer (e.g. a cancer described herein) in a subject, comprising administering to a subject a therapeutically or prophylactically effective amount of a fusion molecule or composition described herein.

[0267] The methods may be effective to reduce the development or progression of a cancer, alleviation of the symptoms of a cancer or reduction in the pathology of a cancer. The methods may be effective to prevent progression of the cancer, e.g. to prevent worsening of, or to slow the rate of development of, the cancer. In some embodiments, the methods may lead to an improvement in the cancer, e.g. a reduction in the symptoms of the cancer or reduction in some other correlate of the severity / activity of the cancer. In some embodiments, the methods may prevent development of the cancer to a later stage (e.g. a chronic stage or metastasis).

[0268] As used herein, a ‘cancer’ may be or comprise any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor. The cancer may be benign or malignant. The cancer may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. The cancer may be of tissues / cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, biliary tract, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells.

[0269] Tumors to be treated may be nervous or non-nervous system tumors. Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous system cancers / tumors may originate in any other non-nervous tissue; examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), hematologic cancer and sarcoma.

[0270] In some embodiments, the cancer is small cell lung cancer (SCLC), neuroendocrine cancer, neuroendocrine cancer variants (e.g. NEPC, LCNEC, NEBC), prostate cancers, breast cancers, gastric cancer, pancreatic cancer, liver cancer, lymphoma, leukaemia, myeloma, brain tumors, skin cancer, uterine cancer, ovarian cancer, or kidney cancer.

[0271] NEPC refers to neuroendocrine prostate carcinoma. LCNEC refers to large cell neuroendocrine carcinoma. NEBC refers to neuroendocrine breast carcinoma.

[0272] In some embodiments, the cancer is small cell lung cancer (SCLC). SCLC is an aggressive neuroendocrine tumor characterized by early metastasis, accounting for 10%— 15% of all lung cancers and associated with poor 5-year survival rates. Treatment options for SCLC remain limited, with most patients experiencing relapse and resistance after radiotherapy, Etoposide and / or platinum-based chemotherapy (e.g. as reviewed in Gazdar et al., Nat Rev Cancer. (2017) 17(12):725-737, which is hereby incorporated by reference in its entirety).

[0273] In some embodiments, the cancer to be treated / prevented comprises cells expressing a tumor antigen which is specifically recognised by the tumor antigen-binding moiety of the fusion molecules disclosed herein. By way of example, in some embodiments the fusion molecule comprises a tumor antigen-binding moiety that specifically recognises DLL3, and the cancer to be treated / prevented is a cancer which is positive for DLL3. By way of further example, in some embodiments the fusion molecule comprises a tumor antigen-binding moiety that specifically recognises EpCAM, and the cancer to be treated / prevented is a cancer which is positive for EpCAM. It will be appreciated that the cancer to be treated is not particularly limited and may be any cancer which expresses a tumor antigen which is recognised by the tumor antigen-binding moiety of the fusion molecules disclosed herein. In some embodiments, the cancer to be treated / prevented comprises cells expressing an antigen selected from: CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: fibroblast activation protein (FAP), B-cell maturation antigen (BCMA), CD19, HER2 / neu, HER3, BING-4, calcium-activated chloride channel 2, cyclin-B1 , 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP-1 , survivin, delta-like ligand 3 (DLL3), tumor-associated calcium signal transducer 2 (TROP2), epidermal growth factor receptor (EGFR), mesenchymal-epithelial transition factor (cMET), B7-H3, B7-H4, PKMYT1 , CEACAM-5, Fra, CLL-1 , GPC3, Six transmembrane epithelial antigen of the prostate 1 (STEAP1), Six transmembrane epithelial antigen of the prostate 2 (STEAP2), Mucin 1 (MUC1), Mucin 16 (MUC16), Claudin family proteins (e.g. CLDN6, CLDN18.2), SEZ6, KLK2, Mesothelin, CD70, EphAIO, CTGF, Nectin-4, AXL, Liv-1 , SLC34A2; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1 , PRAME, SSX-2; lineage restricted antigens: MARTI , Gp100, tyrosinase, TRP-1 / 2, MC1 R, prostate specific antigen, prostate-specific membrane antigen (PSMA); mutated antigens: p-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, MART- 2, p53, Ras, TGF-pRII, FGFR1 , FGFR2, FGFR3, FGFR4; post-translationally altered antigens: MUC1 , idiotypic antigens: Ig, TCR, heat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose- regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B.

[0274] In some embodiments, the cancer to be treated / prevented comprises cells expressing DLL3, EpCAM, TROP2, SEZ6, KLK2, PSMA, STEAP1 , STEAP2, CLDN6 and MUC16.

[0275] In some embodiments, the cancer comprises cells that overexpress DLL3. In some embodiments, the cancer comprises cells that overexpress EpCAM. In some embodiments, the cancer comprises cells that overexpress TROP2. In some embodiments, the cancer comprises cells that overexpress SEZ6. In some embodiments, the cancer comprises cells that overexpress KLK2. In some embodiments, the cancer comprises cells that overexpress PSMA. In some embodiments, the cancer comprises cells that overexpress STEAP1 . In some embodiments, the cancer comprises cells that overexpress STEAP2. In some embodiments, the cancer comprises cells that overexpress CLDN6. In some embodiments, the cancer comprises cells that overexpress MUC16 Overexpression can be determined by detection of a level of expression which is greater than the level of expression by equivalent non-cancerous cells / non- tumor tissue.

[0276] Expression may be determined by any suitable means. Expression may be gene expression or protein expression. Gene expression can be determined e.g. by detection of mRNA encoding DLL3, for example by quantitative real-time PCR (qRT-PCR). Protein expression can be determined e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA. Administration

[0277] Administration of the articles of the present disclosure is preferably in a "therapeutically effective” or “prophylactically effective” amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease / condition and the particular fusion molecule administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0278] Administration of the fusion molecules of the present disclosure may be parenteral, systemic, intravenous, intra-arterial, intramuscular, intracavitary, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, topical or transdermal. Administration may be by injection or infusion. Administration of the fusion molecules of the present disclosure may be intratumoral. In some cases, the articles of the present disclosure may be formulated for targeted delivery to specific cells, a tissue, an organ and / or a tumor.

[0279] Subjects

[0280] The subject in accordance with aspects described herein may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient. A subject may have been diagnosed with a disease or condition requiring treatment (e.g. cancer), may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition.

[0281] In some embodiments, the subject to be treated according to a therapeutic or prophylactic method of the present disclosure herein is a subject having, or at risk of developing, cancer.

[0282] Compositions

[0283] The present disclosure provides a composition comprising a fusion molecule according to the present disclosure.

[0284] The fusion molecules described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. Thus, the present disclosure provides a pharmaceutical composition / medicament comprising a fusion molecule described herein.

[0285] The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0286] Compositions may be formulated for topical, parenteral, systemic, intravenous, intra-arterial, intramuscular, intracavitary, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, topical or transdermal routes of administration. In some embodiments, a pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or administration by ingestion. In some embodiments, compositions comprising a fusion molecule according to the present disclosure are formulated for administration by injection.

[0287] Suitable formulations may comprise the relevant article in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.

[0288] Polynucleotides

[0289] Aspects and embodiments of the present disclosure relate to polynucleotides. The polynucleotides or plurality of polynucleotides described herein encode constituent components of fusion molecules and may be useful in methods of producing fusion molecules. Polynucleotides described herein may find further utilisation in therapeutic methods as described herein. That is, the polynucleotide is employed as a gene therapy for inducing expression of the fusion molecule in the subject to which it is administered.

[0290] A 'polynucleotide' refers to a polymer chain of a plurality of nucleotide monomers linked by bonds between the monomers, typically phosphodiester bonds (e.g. in the case of polynucleotides formed by naturally-occurring nucleotide monomers). Polynucleotides include oligonucleotides, which generally comprise <50 nucleotides. A polynucleotide may be single-stranded, or may be double-stranded ( / .e. may comprise a duplex formed by hydrogen-bonding between complementary nucleotides). Polynucleotides according to the present disclosure may comprise or consist of: single-stranded DNA, double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single-stranded RNA, doublestranded RNA, RNA that is mixture of single- and double-stranded regions, single-stranded molecules comprising DNA and RNA, double-stranded molecules comprising DNA and RNA, and molecules comprising DNA and RNA having a mixture of single- and double-stranded regions.

[0291] Disclosed herein is a polynucleotide encoding any of the fusion molecules disclosed herein comprising an immune cell engager and a cytokine. In some embodiments, a polynucleotide as disclosed herein encodes a single transcript encoding all domains of a fusion molecule as described herein (e.g. a polynucleotide encoding a single transcript encoding a single multi-domain polypeptide).

[0292] In some embodiments, a fusion molecule according to the present disclosure may be encoded by multiple polynucleotides (“a plurality of polynucleotides”). For example, when a fusion molecule according to the present disclosure comprises, or consists of, a polypeptide complex, the constituent polypeptides in the polypeptide complex may be encoded by multiple polynucleotides. In some embodiments, constituent polypeptides of a fusion molecule according to the present disclosure are encoded by nucleotide sequences provided in the same reading frame. In some embodiments, the polynucleotide or plurality of polynucleotides encodes a fusion molecule or constituent polypeptides of a fusion molecule.

[0293] In some embodiments, a polynucleotide as disclosed herein encodes a tumor antigen-binding moiety, an immune cell-binding moiety, and a CH2-CH3 region. It will be appreciated that a linker may be present between one or more of the regions of the polypeptide described herein. In such cases, the polynucleotide as disclosed herein encodes said linker(s). In some embodiments, a polynucleotide as disclosed herein encodes from 5’ to 3’: (i) a tumor antigen-binding moiety, (ii) an immune cell-binding moiety, and (iii) a CH2-CH3 region. In some embodiments, a polynucleotide as disclosed herein encodes from 5’ to 3’: (i) a tumor antigen-binding moiety, (ii) an CD3-binding moiety, and (iii) a CH2-CH3 region. In some embodiments, a polynucleotide as disclosed herein encodes from 5’ to 3’: (i) a DLL3-binding moiety, (ii) an CD3-binding moiety, and (iii) a CH2-CH3 region. In some embodiments, a polynucleotide as disclosed herein encodes from 5’ to 3’: (i) a tumor antigen-binding moiety, (ii) a CH2-CH3 region, and (iii) an immune cell-binding moiety. In some embodiments, a polynucleotide as disclosed herein encodes from 5’ to 3’: (i) a tumor antigen-binding moiety, (ii) a CH2-CH3 region, and (iii) an CD3-binding moiety. In some embodiments, a polynucleotide as disclosed herein encodes from 5’ to 3’: (i) a DLL3-binding moiety, (ii) a CH2-CH3 region, and (iii) an CD3-binding moiety.

[0294] In some embodiments, a polynucleotide as disclosed herein encodes a CH2-CH3 region and a cytokine (e.g. IL-15, Sushi-IL-15 or IL-15-Sushi as described hereinabove, e.g. comprising, or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs: 7, 16, 17, 18, 19 or 20 as described hereinabove). In some embodiments, a polynucleotide as disclosed herein encodes from 5’ to 3’: (i) a CH2-CH3 region and (ii) a cytokine.

[0295] In some preferred embodiments, a plurality of polynucleotides as disclosed herein comprises (i) a first polynucleotide encoding a tumor antigen-binding moiety, an immune cell-binding moiety, and a CH2-CH3 region, and (ii) a second polynucleotide encoding a CH2-CH3 region, and a cytokine.

[0296] For example, a plurality of polynucleotides may comprise (i) a first polynucleotide encoding a tumor antigen-binding moiety, a CD3-binding moiety, and a CH2-CH3 region, and (ii) a second polynucleotide encoding a CH2-CH3 region, and a cytokine.

[0297] In some embodiments, the plurality of polynucleotides comprises: a first polynucleotide encoding from 5’ to 3’:

[0298] (i) a tumor antigen-binding moiety,

[0299] (ii) an immune cell-binding moiety, and

[0300] (iii) a CH2-CH3 region; and a second polynucleotide encoding from 5’ to 3’:

[0301] (i) a CH2-CH3 region, and

[0302] (ii) a cytokine.

[0303] In some embodiments, the plurality of polynucleotides comprises: a first polynucleotide encoding from 5’ to 3’:

[0304] (i) an immune cell-binding moiety,

[0305] (ii) a tumor antigen-binding moiety, and

[0306] (iii) a CH2-CH3 region; and a second polynucleotide encoding from 5’ to 3’:

[0307] (i) a CH2-CH3 region, and

[0308] (ii) a cytokine.

[0309] In some embodiments, a polynucleotide as disclosed herein encodes from 5’ to 3’: (i) a tumor antigenbinding moiety, (ii) a CH2-CH3 region, and (iii) an immune cell-binding moiety.

[0310] In some embodiments, a plurality of polynucleotides comprises: a first polynucleotide encoding from 5’ to 3’:

[0311] (i) a tumor antigen-binding moiety,

[0312] (ii) a CH2-CH3 region, and

[0313] (iii) an immune cell-binding moiety, and a second polynucleotide encoding from 5’ to 3’:

[0314] (i) a CH2-CH3 region, and

[0315] (ii) a cytokine.

[0316] In any of the embodiments described above, the CH2-CH3 region encoded by the first or the second polynucleotide optionally comprise a modification to promote heterodimerisation, to reduce / prevent ADCC / CDC, and / or to extend the serum half-life (e.g. the LALAPG, YTE and ‘knob-into-hole’ modifications described hereinabove).

[0317] In some embodiments, the polynucleotides described herein comprise a signal peptide for protein secretion. A signal peptide is also known as a leader sequence or signal sequence. Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides. Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt and Ensembl, and / or can be identified / predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176). The signal peptide may be present at the N-terminus of the fusion molecule, and may be present in the newly synthesized fusion molecule. The signal peptide provides for efficient trafficking of the fusion molecule. Signal peptides are often removed by cleavage, and thus are not comprised in the mature fusion molecule.

[0318] The polynucleotides of the present disclosure comprise a start codon 5’ to ( / .e. upstream of, in the context of the nucleotide sequence of the polynucleotide) the nucleotide sequence encoding a polypeptide of interest. The start codon is preferably the trinucleotide ‘ATG’.

[0319] In some embodiments, the polynucleotides further comprise a promoter sequence. The promoter sequence is preferably 5’ to the first nucleotide sequence. In some embodiments, the polynucleotide further comprises one or more enhancer sequences. The one or more enhancer sequences are preferably 5’ to the first nucleotide sequence.

[0320] In some embodiments, the polynucleotides further comprise a stop codon. The stop codon is preferably provided immediately 3’ to ( / .e. downstream of, in the context of the nucleotide sequence of the polynucleotide) the trinucleotide encoding the terminal amino acid of the polypeptide encoded by the fifth or sixth nucleotide sequence.

[0321] Vectors

[0322] In some embodiments, the polynucleotides may be, or may be comprised / contained in, a vector, or a plurality of vectors. A ‘vector’ as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell.

[0323] The vector may facilitate delivery of the polynucleotide(s) encoding a polypeptide according to the present disclosure to a cell. The vector may be an expression vector comprising elements required for expressing a polypeptide according to the present disclosure. The vector may comprise elements facilitating integration of the polynucleotide(s) into the genomic DNA of the cell into which the vector is introduced.

[0324] A vector may facilitate delivery of polynucleotides according to the present disclosure to a cell. A vector may be an expression vector, comprising elements required for producing the fusion molecules according to the present disclosure. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0325] The term “operably linked” may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and / or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript may then be translated into a desired pe ptid e (s) / po ly pe ptid e (s) .

[0326] Vectors contemplated in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g. conjugative plasmids (e.g. F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine leukemia virus (MLV)-derived vectors, e.g. SFG vector), lentiviral vectors, adenovirus vectors, adeno- associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus et al., Annu Rev Immunol. (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, which are both hereby incorporated by reference in their entirety.

[0327] In some embodiments, the vector may be a eukaryotic vector, i.e. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0328] Cells

[0329] Aspects and embodiments of the present disclosure relate to cells comprising or expressing a fusion molecule according to the present disclosure. Also provided are cells comprising polynucleotides or vectors according to the present disclosure. A cell according to the present disclosure may comprise a vector, or plurality of vectors, comprising the plurality of polynucleotides according to the present disclosure. A cell according to the present disclosure may comprise a polynucleotide encoding a fusion molecule or encoding constituent polypeptides of a fusion molecule. A cell according to the present disclosure may comprise a polynucleotide encoding a fusion molecule, wherein the fusion molecule comprises an immune cell engager and a cytokine.

[0330] It will be appreciated that where cells are referred to herein in the singular (i.e. ‘a / the cell’), pluralities / populations of such cells are also contemplated.

[0331] The cells disclosed herein may be in vitro and / or may be isolated from the human or animal body.

[0332] A cell according to the present disclosure may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgus, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate).

[0333] The present disclosure also provides a method for producing a cell expressing / comprising a fusion molecule according to the present disclosure, comprising introducing a polynucleotide or vector according to the present disclosure into a cell. In some embodiments, the methods additionally comprise culturing the cell under conditions suitable for expression of the polynucleotide / vector by the cell. In some embodiments, the methods are performed in vitro.

[0334] For recombinant production of fusion molecules according to the present disclosure, any cell suitable for the expression of polypeptides may be used. The cell may be a prokaryote or eukaryote. In some embodiments the cell is a prokaryotic cell, such as a cell of archaea or bacteria. In some embodiments the bacteria may be Gram-negative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli. In some embodiments, the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e.g. a cell described hereinabove.

[0335] In some embodiments, the cell is, or is derived from, a cell type commonly used for the expression of polypeptides for use in therapy in humans. Exemplary cells are described e.g. in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461 (hereby incorporated by reference in its entirety), and include e.g. CHO, HEK 293, PER.C6, NSO and BHK cells.

[0336] Kits

[0337] The present disclosure also provides kits of parts. A kit according to the present disclosure may comprise components for performing a method described herein, in whole or in part.

[0338] The kit may have at least one container having a predetermined quantity of a fusion molecule or composition described herein.

[0339] In some aspects of the present disclosure a kit of parts is provided. In some embodiments, the kit may comprise a fusion molecule or composition described herein, and which may be provided in a predetermined quantity.

[0340] The kit may provide a fusion molecule or composition described herein together with instructions for administration to a patient in order to treat a specified disease / condition (e.g. a disease / condition described herein, e.g. cancer) e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.

[0341] The kit may further comprise reagents, buffers and / or standards required for execution of a method according to the present disclosure.

[0342] Sequence identity

[0343] As used herein, ‘sequence identity’ refers to the percent of nucleotides / amino acid residues in a subject sequence that are identical to nucleotides / amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J., Bioinformatics (2005) 21 , 951-960), T-coffee (Notredame etal., J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer, BMC Bioinformatics (2005) 6,298) and MAFFT (Katoh and Standley, Molecular Biology and Evolution (2013) 30(4) 772-780) software.

[0344] When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0345] Sequences

[0346] ***

[0347] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0348] The section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described.

[0349] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0350] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0351] As used herein, a ‘peptide’ refers to a chain of two or more amino acid monomers linked by peptide bonds. A peptide typically has a length in the region of about 2 to 50 amino acids. A ‘polypeptide’ is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids. Reference herein to peptides, polypeptides and proteins also includes glycopeptides / glycopolypeptides / glycoproteins, lipopeptides / lipopolypeptides / lipoproteins, nucleopeptides / nucleopolypeptides / nucleoproteins, etc.

[0352] As used herein, an amino acid sequence, or a region of a polypeptide, which ‘corresponds’ to a specified reference amino acid sequence or region of a polypeptide has at least 60%, e.g. one of at least >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the amino acid sequence of the amino acid sequence / polypeptide / region. An amino acid sequence / region / position of a polypeptide / amino acid sequence which ‘corresponds’ to a specified reference amino acid sequence / region / position of a polypeptide / amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951-960).

[0353] Similarly, a nucleotide sequence, or a region of a polynucleotide, which ‘corresponds’ to a specified reference nucleotide sequence or region of a polynucleotide has at least 60%, e.g. one of at least >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the amino acid sequence of the nucleotide sequence / polynucleotide / region. A polynucleotide / region / position of a polynucleotide / nucleotide sequence which ‘corresponds’ to a specified reference nucleotide sequence / region / position of a polynucleotide / nucleotide sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951-960). As used herein, an amino acid sequence (e.g. the amino acid sequence of a peptide / polypeptide / domain / region) which is ‘derived from’ a reference amino acid sequence (e.g. the amino acid sequence of a reference peptide / polypeptide / domain / region) comprises, or consists of, an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference amino acid sequence. Similarly, a nucleotide sequence (e.g. a nucleotide sequence of a polynucleotide) which is ‘derived from’ a reference nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference nucleotide sequence.

[0354] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.

[0355] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0356] Methods described herein may preferably be performed in vitro. The term ‘in vitro’ is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with / on intact multi-cellular organisms.

[0357] Brief Description of the Figures

[0358] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures.

[0359] Figure 1. Structure of exemplary engagers. 1A. #2 Tarlatamab (Benchmark) (anti-DLL3 scFv and anti- CD3 scFv fused to single chain Fc (ScFc)). 1 B. #7 TriEngager using anti-DLL3 scFv and anti-CD3 scFv and Knob-into-hole (hetero Fc). 1C. #8 QuadEngager using anti-DLL3 scFv and anti-CD3 scFv, hetero Fc and IL-15 fusion. 1 D. #9 QuadEngager using anti-DLL3 scFv and anti-CD3 scFv, hetero Fc and Sushi-IL- 15 fusion. 1E. #10 QuadEngager using anti-DLL3 scFv and anti-CD3 scFv, hetero Fc and Neo-2 / 15 fusion. 1F. #11 TriEngager using anti-DLL3 scFv and anti-CD3 scFv on the same arm of the hetero Fc. 1G. #12 QuadEngager using anti-DLL3 scFv and anti-CD3 scFv on the same arm of the hetero Fc and IL- 15 fusion. 1H. #13 QuadEngager using anti-DLL3 scFv and anti-CD3 scFv on the same arm of the hetero Fc and Sushi-IL-15 fusion. 11. #15 HPN328 (Benchmark) (anti-DLL3 VH and anti-CD3 scFv fused to antialbumin). 1 J. #38 TriEngager using anti-DLL3 VH, hetero Fc and anti-CD3 scFv. 1K. #39 QuadEngager using anti-DLL3 VH, hetero Fc, anti-CD3 scFv and IL-15 fusion using GGGGSGGGGSGGGGS linker. 1L. #40 QuadEngager using anti-DLL3 VH, hetero Fc, anti-CD3 scFv and IL-15 fusion using GGGGSGGGGSGGGGSGGGGS linker. Figure 2. Binding of #2 Tarlatamab, #7 TriEngager, #8 QuadEngager and #9 QuadEngager to SHP-77 cancer cells (high DLL3 expression level) (2A) and NCI-H82 cancer cells (medium DLL3 expression level) (2B). gMFI = Geometric mean fluorescence intensity.

[0360] Figure 3. T cell activation data (NFAT-GFP) for #2 Tarlatamab, #7 TriEngager, #8 QuadEngager and #9 QuadEngager in SHP-77 cancer cells (high DLL3 expression level) (3A), NCI-H82 cancer cells (medium DLL3 expression level) (3B) and NCI-H460 cells (no DLL3 expression) (3C). gMFI = Geometric mean fluorescence intensity.

[0361] Figure 4. T cell activation data (NFAT-GFP). 4A. #2 Tarlatamab, #7 TriEngager, #8 QuadEngager, #9 QuadEngager and #10 QuadEngager in SHP-77 cancer cells. 4B. #2 Tarlatamab, #11 TriEngager, #12 QuadEngager and #13 QuadEngager in SHP-77 cancer cells. gMFI = Geometric mean fluorescence intensity.

[0362] Figure 5. CD3 and DLL3 binding affinity. 5A. CD3 binding affinity for #2 Tarlatamab (Benchmark), #7 TriEngager, #8 QuadEngager, #9 QuadEngager, #10 QuadEngager, #11 TriEngager, #12 QuadEngager and #13 QuadEngager. 5B. DLL3 binding affinity for #2 Tarlatamab (Benchmark), #7 TriEngager, #8 QuadEngager, #9 QuadEngager, #10 QuadEngager, #11 TriEngager, #12 QuadEngager and #13 QuadEngager.

[0363] Figure 6. T-cell dependent cellular cytotoxicity (TDCC) in SHP-77 cancer cells (Effector:Target=2:1) for #2 Tarlatamab (Benchmark), #7 TriEngager, #7 TriEngager + IL-15 combination, #8 QuadEngager, #9 QuadEngager. 6A. TDCC at 24 hours, 48 hours and 96 hours 6B. TDCC with addition of cancer cells; cytotoxicity at 96 hours (cancer cells added at 48 hours) and 144 hours (cancer cells added at 48 and 96 hours).

[0364] Figure 7. T-cell dependent cellular cytotoxicity (TDCC) in SHP-77 cancer cells. Effector:Target ratio=2:1. 7A. Comparison of TDCC for #2 Tarlatamab (Benchmark), 11-15 alone, #7 TriEngager, and #7 TriEngager + IL-15 combination. 7B. TDCC for #2 Tarlatamab, #11 TriEngager, #11 TriEngager + IL-15 combination, #12 QuadEngager and #13 QuadEngager.

[0365] Figure 8. T-cell dependent cellular cytotoxicity (TDCC) in SHP-77 cancer cells. Effector:Target ratio= 1 :1 . 8A. Comparison of TDCC for #2 Tarlatamab, #11 TriEngager, #11 TriEngager + IL-15 combination, #12 QuadEngager and #13 QuadEngager at 24, 48 and 96 hours 8B. TDCC with addition of cancer cells; cytotoxicity at 96 hours (cancer cells added at 48 hours).

[0366] Figure 9. Cytokine release data. Condition: Effector:Target ratio=2:1 (Effector: PBMCs, Target: luciferase SHP-77). 9A. Cytokine production (IL-6, TNF-a, IFN-y, IL-10) in PBMCs induced by #2 Tarlatamab, #7 TriEngager and #7 TriEngager +IL-15 combination. 9B. Production of cytosolic components (granzyme A, granzyme B, perforin, granulysin) induced by #2 Tarlatamab, #7 TriEngager and #7 TriEngager +IL-15 combination. Figure 10. Cytokine release data. Condition: E:T ratio=2:1 (Effector: PBMCs, Target: luciferase SHP-77). 10A. Cytokine production (IL-6, TNF-a, IFN-y, IL-10) in PBMCs induced by IL-15, #2 Tarlatamab, #11 TriEngager, #11 TriEngager +IL-15 combination, #12 QuadEngager and #13 QuadEngager. 10B.

[0367] Production of cytosolic components (granzyme A, granzyme B, perforin, granulysin) induced by IL-15, #2 Tarlatamab, #11 TriEngager, #11 TriEngager +IL-15 combination, #12 QuadEngager and #13 QuadEngager.

[0368] Figure 11. T-cell dependent cellular cytotoxicity (TDCC) in SHP-77 cancer cells. Effector:Target ratio= 1 :1 . Comparison of TDCC for #15 HPN328 (Benchmark), #38 TriEngager, #38 TriEngager + IL-15 combination, #39 QuadEngager and #40 QuadEngager at 24 hours, 48 hours and 96 hours.

[0369] Figure 12. T-cell dependent cellular cytotoxicity (TDCC) in SHP-77 cancer cells. Effector:Target ratio=0.5:1. Comparison of TDCC for pTCE20 (Benchmark), pTCE294 / 297.

[0370] Figure 13. T-cell mediated killing of a DLL3 expressing cancer cell line under challenge conditions (according to Example 3.2). (a) T-cell dependent cellular cytotoxicity (TDCC) in SHP-77 cancer cells. Effector:Target ratio=1 :1. Comparison of TDCC for pTCE20 (Benchmark), pTCE333 / 334. (b) T-cell dependent cellular cytotoxicity (TDCC) in SHP-77 cancer cells. Effector:Target ratio=1 :1 . Comparison of TDCC for pTCE20 (Benchmark), TCE344 / 345 and TCE344 / 346.

[0371] Figure 14. T-cell dependent cellular cytotoxicity (TDCC) in SHP-77 cancer cells. Effector:Target ratio=1 :1. Comparison of TDCC for pTCE20 (Benchmark), pTCE174 / 98, and pTCE80 / 73. Data produced from multi-day repeated tumor cell challenge assay (as described in Example 3.4).

[0372] Figure 15. (a) T-cell dependent cellular cytotoxicity (TDCC) in SHP-77 cancer cells. Effector:Target ratio=1 :1. Comparison of TDCC for pTCE20 (Benchmark), pTCE73 / 80, and pTCE333 / 334. Data produced from challenge assay (according to the methods of Example 3.3. (b) T-cell dependent cellular cytotoxicity (TDCC) in SHP-77 cancer cells. Effector:Target ratio=0.25:1 . Comparison of TDCC for pTCE20 (Benchmark), pTCE73 / 80, pTCE333 / 334, and pTCE344 / 345. Data produced from challenge assay (according to the methods of Example 3.4).

[0373] Figure 16. Diagram of exemplary K shape QuadEngager.

[0374] Figure 17. Diagram of exemplary K shape QuadEngager.

[0375] Figure 18. Diagram of exemplary K shape QuadEngager.

[0376] Examples

[0377] Example 1 : Materials and Methods 1.1 Engager production and purification

[0378] To produce the engager, ExpiCHO-S cells (Gibco) were co-transfected with heterodimeric Fc plasmids at a 1 :1 ratio, according to the manufacturer's instructions. After 8 days, the supernatant was collected and purified using a HiTrap Protein G column (Cytiva, 17040401). Protein purity was then assessed and / or further purified by size-exclusion chromatography using a Superdex 200 Increase column (GE Healthcare).

[0379] 1 .2 Binding Assays

[0380] CD3 binding assay

[0381] Recombinant His-tagged CD3 epsilon (SinoBiological, 10977-H08H) was immobilized on high proteinbinding 96-well ELISA plates (Thermo, #442404) at a concentration of 1 pg / ml in phosphate-buffered saline (PBS, PROTECH, #BF205-1 L) overnight at 4°C. The plates were then blocked with PBS containing 3% bovine serum albumin (OmicsBio, #AD0023) at room temperature for 1 hour, followed by washing with PBS containing 0.05% Tween 20 (PBST) (Merck, #8.22184.0500). Recombinant proteins were prepared as 3-fold serial dilutions starting at 10000 ng / ml in PBS with 3% bovine serum albumin and incubated for 2 hours at 37°C. After washes with PBST, a 1 :20000 dilution of an Anti-Human IgG Fc- Peroxidase antibody in PBS with 3% bovine serum albumin was added to the plates and incubated for 1 hour at 37°C, followed by another wash with PBST. Protein binding was detected using TMB substrate (Invitrogen, #002023), and absorbance was measured at 450 nm.

[0382] DLL3 binding assay

[0383] Recombinant His-tagged DLL3 (Abeam, ab255797) was immobilized on high protein-binding 96-well ELISA plates (Thermo, #442404) at a concentration of 1 pg / ml in phosphate-buffered saline (PBS, PROTECH, #BF205-1 L) overnight at 4°C. The plates were then blocked with PBS containing 3% bovine serum albumin (OmicsBio, #AD0023) at room temperature for 1 hour, followed by washing with PBS containing 0.05% Tween 20 (PBST) (Merck, #8.22184.0500). Recombinant proteins were prepared as 3- fold serial dilutions starting at 10000 ng / ml in PBS with 3% bovine serum albumin and incubated for 2 hours at 37°C. After washes with PBST, a 1 :20000 dilution of an Anti-Human IgG Fc-Peroxidase antibody in PBS with 3% bovine serum albumin was added to the plates and incubated for 1 hour at 37°C, followed by another wash with PBST. Protein binding was detected using TMB substrate (Invitrogen, #002023), and absorbance was measured at 450 nm.

[0384] 1 .3 T Cell Activation Assay

[0385] T cell activation was evaluated by Jurkat NFAT-GFP reporter cells co-cultured with SHP-77 cancer cells (high DLL3 expression level), NCI-H82 cancer cells (medium DLL3 expression level) and NCI-H460 cells (no DLL3 expression) at a ratio of 1 :1 . Geometric mean fluorescence intensity (gMFI) of green fluorescent protein (GFP) reporter gene driven by nuclear factor of activated T cells (NFAT) activation via TCR signaling was measured by flow cytometry.

[0386] 1 .4 T-cell dependent cellular cytotoxicity (TDCC)

[0387] T-cell dependent cellular cytotoxicity (TDCC) was measured in SHP-77 cells using effector target ratios of 2:1 (Effector: PBMCs, Target: luciferase SHP-77), 1 :1 , 0.5:1 , and 0.25:1. Luciferase SHP-77 cells were plated at 10,000 cells per well in U-bottom 96 well plate and co-cultured with PBMCs at the indicated E:T ratios. Engagers were added at a final concentration of 10,000 pM, with an 8-point titration curve using 3- fold serial dilutions across the plate. Following incubation for 16 to 144 hours, additional cancer cells were introduced at 48 hours, or at both 48 and 96 hours for serial killing measurements. Cell viability was determined using the One-Gio™ Luciferase Assay (Promega #E6120).

[0388] 1 .5 Cytokine Release

[0389] Cytokine and cytolytic component production induced by T-cell engagers (TCE) was assessed under an E:T ratio of 2:1 , using PBMCs as effector cells and luciferase-expressing SHP-77 as target cells. PBMCs and SHP-77 cells were co-incubated with the engagers at the indicated concentrations and time points. After incubation, culture supernatants were collected and diluted 2- to 4-fold. Cytokine levels were measured using the LEGENDplex™ Human CD8 / NK Panel (13-plex) wA / bP V02 (BioLegend, #741187) in accordance with the manufacturer's instructions.

[0390] Example 2: Development of Improved Immune Cell Engagers

[0391] In this work, various multifunctional proteins (TriEngager and QuadEngager constructs) were designed, leveraging their multi-faceted immune cell activation to enhance tumor treatment effectiveness.

[0392] 2.1 Expression of Engagers and Combinations used in this study

[0393] Exemplary engagers were designed, expressed and purified. Engager profiles and combination treatments used in this study are set out in Table 1 .

[0394] Table 1 : Engagers and Combinations used in this study

[0395] #2 Tarlatamab

[0396] Tarlatamab was used as a benchmark. Tarlatamab (also known as AMG 757; DrugBank Acc. No: DB17256) is a HLE BiTE consisting of an anti-DLL3 scFv, an anti-CD3e scFv and an Fc domain. The anti-DLL3 scFv which binds to cancer cells, while the anti-CD3 scFv binds to T cells. The ScFc (single chain Fc) is designed to extend the pharmacokinetics. Since it is already on the market, its cytokine release can serve as a safety benchmark for comparison with other engager designs. The amino acid sequence of Tarlatamab is set out in SEQ ID NO: 33.

[0397] The anti-DLL3 scFv and anti-CD3 scFv were fused with a short linker (GGGGS). Yield before affinity chromatography and FPLC purification: 39 mg / L. Yield after affinity chromatography and FPLC purification: 31.5 mg / L. Purity before FPLC purification: 80.77 %.

[0398] #7 TriEngager

[0399] Both anti-DLL3 scFv and anti-CD3 scFv are sequences from Tarlatamab binders. A Knob-into-hole (hetero Fc) was designed to extend the pharmacokinetics. The amino acid sequence of #7 TriEngager is set out in SEQ ID NO: 34 and SEQ ID NO: 35. A hetero Fc format for engager design can distribute the molecular weight on both sides of the antibody and allows expression of multiple complex moiety as separate polypeptide chains and assembled into one polypeptide complex after expression and folding of individual chain, such design reduce the loading of host cell protein expression and folding machinery and therefore allows incorporation of more functional moiety (i.e. cytokines, tumor cell binders and immune cell binders) to be incorporated into a protein complex, which renders non-producible if all the moieties were fused and produced as a single polypeptide chain. After the hetero proteins are produced separately, they rely on knob-in-hole shape complementarity and disulfide bonds to correctly associate into a complete knob-into-hole antibody. This structure also enables the design of more domains within the same conformation. However, the distance between the DLL3 and CD3 binders on the TriEngager is greater than on Tarlatamab, which has the possibility of reducing its ability to activate T cells. TriEngager has a higher protein yield compared to Tarlatamab. Yield before affinity chromatography and FPLC purification: 113.99 mg / L. Yield after affinity chromatography and FPLC purification: 91.6 mg / L. Purity before FPLC purification: 80.4 %.

[0400] #7 TriEngager + IL-15 combination therapy

[0401] The addition of IL-15 aims to support long-term T cell survival, proliferation, and activation. Moreover, IL- 15 has been found to activate NK cells. It was hypothesized that the dual activation of T cells and NK cells may synergistically enhance tumor killing.

[0402] #8 QuadEngager

[0403] Both anti-DLL3 scFv and anti-CD3 scFv are sequences from Tarlatamab binders. A knob-into-hole (hetero Fc) was designed to extend the pharmacokinetics. The amino acid sequence of #8 QuadEngager is set out in SEQ ID NO: 35 and SEQ ID NO: 36. IL-15 was fused to the #8 QuadEngager with the aim of supporting long-term T cell survival, proliferation, and activation for functionalities such as cancer cell killing, as demonstrated by the TriEngager plus IL-15 combination treatment.

[0404] The protein yield of the #8 QuadEngager is significantly lower than that of the #7 TriEngager. This was thought to be due to the increased complexity of the protein construct from fusion of IL-15 in the #8 QuadEngager, Yield before affinity chromatography and FPLC purification: 49.26 mg / L. Yield after affinity chromatography and FPLC purification: 34.74 mg / L. Purity before FPLC purification: 70.53 %.

[0405] #9 QuadEngager

[0406] Both anti-DLL3 scFv and anti-CD3 scFv are sequences from Tarlatamab binders. A knob-into-hole (hetero Fc) is designed to extend the pharmacokinetics. The amino acid sequence of #9 QuadEngager is set out in SEQ ID NO: 35 and SEQ ID NO: 37. Fusing Sushi-IL-15 to the #9 QuadEngager aims to support long-term T cell survival, proliferation, and activation for functionalities such as cancer cell killing, as demonstrated by the #7 TriEngager plus IL-15 combination treatment.

[0407] The protein yield of the #9 QuadEngager is significantly lower than that of the #7 TriEngager and #8 QuadEngager. This was thought to be due to the increased complexity of the protein construct from fusion of Sushi-IL-15 in the #9 QuadEngager. Yield before affinity chromatography and FPLC purification: 71 .33 mg / L. Yield after affinity chromatography and FPLC purification: 30.9 mg / L. Purity before FPLC purification: 43.32 %.

[0408] #10 QuadEngager

[0409] Both anti-DLL3 scFv and anti-CD3 scFv are sequences from Tarlatamab binders. A knob-into-hole (hetero Fc) is designed to extend the pharmacokinetics. Fusion of Neo-2 / 15 in the #10 QuadEngager aims to support long-term T cell survival, proliferation, and activation for functionalities such as cancer cell killing, as demonstrated by the #7 TriEngager plus IL-15 combination treatment. The amino acid sequence of #10 QuadEngager is set out in SEQ ID NO: 35 and SEQ ID NO: 38. #11 TriEngager

[0410] Both anti-DLL3 scFv and anti-CD3 scFv are sequences from Tarlatamab binders. A knob-into-hole (hetero Fc) is designed to extend the pharmacokinetics. The linker between anti-DLL3 scFv and anti-CD3 scFv is amino acid SGGGGS, which is as same as the linker on #2 Tarlatamab between these two binders. The amino acid sequence of #11 TriEngager is set out in SEQ ID NO: 3 and SEQ ID NO: 39. #11 TriEngager has similar biological functions compared to #2 Tarlatamab.

[0411] This TriEngager has similar protein yield compared to #2 Tarlatamab. Yield before affinity chromatography and FPLC purification: 96.13 mg / L. Yield after affinity chromatography and FPLC purification: 42.67 mg / L. Purity before FPLC purification: 44.39 %.

[0412] #11 TriEngager + IL-15 combination therapy

[0413] The addition of IL-15 aims to support long-term T cell survival, proliferation, and activation for functionalities such as cancer cell killing. Moreover, IL-15 has been found to activate NK cells. It was hypothesized that the dual activation of T cells and NK cells may synergistically enhance tumor killing.

[0414] #12 QuadEngager

[0415] Both anti-DLL3 scFv and anti-CD3 scFv are sequences from Tarlatamab binders. A knob-into-hole (hetero Fc) is designed to extend the pharmacokinetics. The linker between anti-DLL3 scFv and anti-CD3 scFv is amino acid SGGGGS, which is as same as the linker on #2 Tarlatamab between these two binders. Fusion of IL-15 in the #12 QuadEngager aims to support long-term T cell survival, proliferation, and activation for functionalities such as cancer cell killing, as demonstrated by the #11 TriEngager plus IL-15 combination treatment. The amino acid sequence of #12 QuadEngager is set out in SEQ ID NO: 39 and SEQ ID NO: 40.

[0416] The protein yield of the #12 QuadEngager is lower than that of the #11 TriEngager. Yield before affinity chromatography and FPLC purification: 26.71 mg / L. Yield after affinity chromatography and FPLC purification: 7.03 mg / L. Purity before FPLC purification: 26.33 %. It was hypothesized that this is due to the increased complexity of the protein construct from fusing IL-15 to the #12 QuadEngager.

[0417] #13 QuadEngager

[0418] Both anti-DLL3 scFv and anti-CD3 scFv are sequences from Tarlatamab binders. A knob-into-hole (hetero Fc) is designed to extend the pharmacokinetics. The linker between anti-DLL3 scFv and anti-CD3 scFv is amino acid GGGGS, which is as same as the linker on #2 Tarlatamab between these two binders. Fusion of Sushi-IL-15 in the #13 QuadEngager aims to support long-term T cell survival, proliferation, and activation for functionalities such as cancer cell killing, as demonstrated by the #11 TriEngager plus IL-15 combination treatment. The amino acid sequence of #13 QuadEngager is set out in SEQ ID NO: 39 and SEQ ID NO: 41.

[0419] The protein yield of the #13 QuadEngager is significantly lower than that of the #11 TriEngager. Yield before affinity chromatography and FPLC purification: 45.15 mg / L. Yield after affinity chromatography and FPLC purification: 16.13 mg / L. Purity before FPLC purification: 35.74 %. It was hypothesized that this is due to the structural complexity of Sushi-IL-15.

[0420] #15 HPN328

[0421] HPN328 was used as a benchmark. HPN328 has anti-DLL3 VHH which binds to cancer cells, while the anti-CD3 scFv binds to T cells. Anti-albumin is designed to extend the pharmacokinetics. The amino acid sequence of #15 HPN328 is set out in SEQ ID NO: 42.

[0422] Yield before affinity chromatography and FPLC purification: 0.771 mg / L. Purity before FPLC purification: 90 %.

[0423] #38 TriEngager

[0424] Both the anti-DLL3 VHH and anti-CD3 ScFv are sequences from HPN328 binders. A knob-into-hole (hetero Fc) is designed to extend the pharmacokinetics. The amino acid sequence of #38 TriEngager is set out in SEQ ID NO: 3 and SEQ ID NO: 43.

[0425] Yield before affinity chromatography and FPLC purification: 341.2 mg / L. Yield after affinity chromatography and FPLC purification: 202 mg / L. Purity before FPLC purification: 59.21 %.

[0426] #38 TriEngager + IL-15 combination therapy

[0427] The addition of IL-15 aims to support long-term T cell survival, proliferation, and activation for functionalities such as cancer cell killing. Moreover, IL-15 has been found to activate NK cells. It was hypothesized that the dual activation of T cells and NK cells may synergistically enhance tumor killing.

[0428] #39 QuadEngager

[0429] Both the anti-DLL3 VHH and anti-CD3 ScFv are sequences from HPN328 binders. A knob-into-hole (hetero Fc) is designed to extend the pharmacokinetics. Fusing IL-15 to the #39 QuadEngager aims to support long-term T cell survival, proliferation, and activation, as demonstrated by the #38 TriEngager plus IL-15 combination treatment. IL-15 is fused to the Fc knob using three sets of GGGGS linker (15 amino acids) (SEQ ID NO: 24). The amino acid sequence of #39 QuadEngager is set out in SEQ ID NO: 40 and SEQ ID NO: 43.

[0430] Yield before affinity chromatography and FPLC purification: 85.33 mg / L. Yield after affinity chromatography and FPLC purification: 46.9 mg / L. Purity before FPLC purification: 55.03 %.

[0431] #40 QuadEngager

[0432] Both the anti-DLL3 VHH and anti-CD3 ScFv are sequences from HPN328 binders. A knob-into-hole (hetero Fc) is designed to extend the pharmacokinetics. Fusing IL-15 to the #40 QuadEngager aims to support long-term T cell survival, proliferation, and activation for functionalities such as cancer cell killing, as demonstrated by the #38 TriEngager plus IL-15 combination treatment. IL-15 is fused to the Fc knob using four sets of GGGGS linker (20 amino acids) (SEQ ID NO: 25). The amino acid sequence of #40 QuadEngager is set out in SEQ ID NO: 44 and SEQ ID NO: 43.

[0433] Yield before affinity chromatography and FPLC purification: 129.5 mg / L. Yield after affinity chromatography and FPLC purification: 70.11 mg / L. Purity before FPLC purification: 54.14 %.

[0434] 2.2 Binding to SHP-77 (high DLL3 expression level) and NCI-H82 (medium DLL3 expression level) cancer cells

[0435] Tarlatamab binds to both SHP-77 and NCI-H82 cancer cells (Figures 2A and 2B). #7 TriEngager also binds to both SHP-77 and NCI-H82 cancer cells (Figures 2A and 2B).

[0436] The #8 QuadEngager exhibits very weak binding affinity towards SHP-77 and NCI-H82 cancer cells expressing DLL3 (Figures 2A and 2B). It was speculated that the #8 QuadEngager's more intricate conformation, compared to Tarlatamab and #7 TriEngager, may lead to folding issues during protein production, thereby compromising the binder's integrity and binding ability. The #9 QuadEngager also exhibits very weak binding affinity towards SHP-77 and NCI-H82 cancer cells expressing DLL3 (Figures 2A and 2B). It was speculated that similarly to #8 QuadEngager, the complex structure may reduce the integrity during protein production.

[0437] 2.3 T cell Activation data (NFAT-GFP)

[0438] T cell activation by engagers was assessed. Tarlatamab induces higher NFAT GFP signaling activation than other engagers (Figures 3A and 3B). As expected, (NCI-H460 cells do not express DLL3), NFAT GFP activation was not induced when NCI-H460 cells were used in T-cells stimulation assay in the presence of the engagers (Figure 3C).

[0439] The #7 TriEngager has a significantly lower NFAT GFP signaling compared to Tarlatamab especially at high doses (3333-10000 pM) (Figures 3A and 3B). It was hypothesized that this may be due to antibody internalization caused by the different engager conformations, or the greater distance between DLL3 and CD3. The #8 QuadEngager and the #9 QuadEngager do not induce NFAT GFP signaling activation (Figures 3A and 3B). #10 QuadEngager induces higher NFAT GFP signaling than the #8 QuadEngager and the #9 QuadEngager at higher doses (Figure 4A).

[0440] The #11 TriEngager induces similar NFAT GFP signaling compared to Tarlatamab (Figure 4B). The #12 QuadEngager and the #13 QuadEngager also induce NFAT-GFP signaling activation, with their signal only slightly lower than that of #2 Tarlatamab and #11 TriEngager (Figure 4B), suggesting that these formats are effective. These data suggest that the presence of IL-15 or Sushi-IL-15 does not impact protein folding.

[0441] Taken together, ( / .e. combined with the data for the #8 and #9 QuadEngagers (Figure 3A and 3B)) the T cell activation data suggests that based on the complexity of cytokines, the arm with the cytokine should only contain the cytokine itself and should not be fused with the scFv domain. When the cytokine arm contains only the cytokine, it can fold well with the anti-DLL3 / anti-CD3 scFv at the other end without affecting T cell activation function.

[0442] 2.4 Cytokine fusion to anti-DLL3 on the same arm) disrupts anti-DLL3 binding

[0443] To confirm that the relative position of the cytokine and the tumor antigen-binding moiety is important, CD3 and DLL3 binding affinity was tested by ELISA.

[0444] All engagers tested showed CD3 binding affinity (Figure 5A). EC50 values were: #2 Tarlatamab = 63.94 ng / ml, #7 TriEngager = 30.9 ng / ml, #8 QuadEngager = 36 ng / ml, #9 QuadEngager = 47.7 ng / ml, #10 QuadEngager = 37 ng / ml, #11 TriEngager = 52.97 ng / ml, #12 QuadEngager = 40.64 ng / ml, #13 QuadEngager = 92.4 ng / ml.

[0445] However, cytokine fusing to anti-DLL3 on the same Fc arm was found to disrupt DLL3 binding (Figure 5B). QuadEngagers with a cytokine fusion on the same Fc arm as anti-DLL3 showed the lowest binding affinity; EC50: #8 QuadEngager = 3084 ng / ml, #9 QuadEngager = 583.8 ng / ml and #10 QuadEngager = 7121 ng / ml compared to #2 Tarlatamab = 63.97 ng / ml, #7 TriEngager = 43.1 . ng / ml, #11 TriEngager = 75.48 ng / ml, #12 QuadEngager = 55.34 ng / ml and #13 QuadEngager = 119.6 ng / ml.

[0446] These results suggest that the best engager formats have the tumor antigen-binding moiety and the immune cell-binding moiety on the opposite Fc arm to the cytokine.

[0447] 2.5 T-cell dependent cellular cytotoxicity

[0448] T-cell dependent cellular cytotoxicity (TDCC) was tested using an Effector:Target ratio of 2:1 (Effector: PBMCs, Target: luciferase SHP-77). Tarlatamab induces the strongest TDCC effect against tumor cells (Figures 6A and Figure 6B). Even when additional cancer cells are provided for re-stimulation to measure serial-killing (Figure 6B), Tarlatamab-activated T cells still maintain strong TDCC activity at 96 and 144 hours.

[0449] The #7 TriEngager induces lower TDCC effect against tumor cells compared to Tarlatamab (Figure 6A- 6B). From the 96- and 144-hours serial-killing results (addition of cancer cells), it appears that the #7 TriEngager-treated PBMCs gradually lose their cytotoxicity against cancer cells (Figure 6B).

[0450] The #7 TriEngager plus IL-15 combination treatment induces similar TDCC effect against tumor cells compared to Tarlatamab. From the 144 hours serial-killing results (addition of cancer cells), the TriEngager plus IL-15 combination treatment induces higher T cell cytotoxicity against tumor cells (from 123-370 pM) (Figure 6B), suggesting that IL-15 supplementation leads to a long-term tumor suppression effect.

[0451] The #8 QuadEngager induces very low TDCC effect against tumor cells compared to Tarlatamab and other engagers (Figures 6A and Figure 6B). The #9 QuadEngager also induces very low TDCC effect against tumor cells compared to Tarlatamab and other engagers at low protein concentrations (0-1000 pM). However, the #9 QuadEngager led to T cell activation and TDCC effect when administered at high concentrations (1000-10000 pM) (Figures 6A and Figure 6B). It was hypothesized that this is because T cell activity was induced by the Sushi-I L15 component itself. It was subsequently shown that IL-15 alone induced less TDCC than Tarlatamab and #7 TriEngager in combination with IL-15 (Figure 7A).

[0452] The #11 TriEngager exhibits similar cytotoxic effects to #2 Tarlatamab (Figure 7B). The comparison between #7 TriEngager and #11 TriEngager demonstrates that the distance between anti-CD3 and anti- DLL3 influences the biological function of TCE in T cell activation. The #11 TriEngager plus IL-15 combination therapy induces the highest TDCC effect against tumor cells compared to #2 Tarlatamab, #11 TriEngager, and IL-15 treatment group (Figure 7B). The IL-15 only treatment group induces a weak cytotoxic effect against the cancer cells, and the cytotoxicity gradually increases as the incubation time extends (from 40% at 24 hours to 80% at 96 hours).

[0453] The #12 QuadEngager induces higher TDCC effect against tumor cells compared to #2 Tarlatamab and #11 T riEngager treatment group. Additionally, the difference between #12 QuadEngager and #2 Tarlatamab becomes larger compared to when the cell incubation time increased to 96 hours. The #13 QuadEngager has a similar cytotoxicity effect is similar to the #12 QuadEngager (Figure 7B).

[0454] TDCC was also tested using an effector target ratio of 1 :1 (Effector: PBMCs, Target: luciferase SHP-77) (Figure 8). When the E:T ratio was lowered, both #2 Tarlatamab and #11 TriEngager no longer achieve 100% cytotoxicity. #11 TriEngager induces a similar cytotoxic effect on cancer cells compared to #2 Tarlatamab. At the 96-hour time point, especially when additional cancer cells are added, both #2 Tarlatamab and #11 TriEngager were no longer able to effectively kill the cancer cells, reaching only around 70% cytotoxicity at the highest protein treatment dose (Figure 8A).

[0455] The #11 TriEngager plus IL-15 combination therapy reached the highest cytotoxicity compared to other groups only after 48 hours. Additionally, IL-15 alone can no longer effectively induce cytotoxicity against cancer cells, and its cytotoxicity gradually decreases as the cell incubation time increases. It was hypothesized that when T cell density is reduced (low E:T ratio), the cytokine autocrine concentration from IL-15-induced T cell activation is insufficient to achieve a level of T cell activation that would result in significant cytotoxicity.

[0456] Using an effector target ratio of 1 :1 , the #12 QuadEngager and #13 QuadEngager still induce higher TDCC against tumor cells compared to #2 Tarlatamab and #11 TriEngager treatment groups (Figure 8A and 8B). Furthermore, this difference Tarlatamab becomes more distinct when the ET ratio is 1 :1 .

[0457] 2.6 Cytokine Release

[0458] Production of cytokines and cytolytic components in PBMCs was measured to assess safety of engagers. Tarlatamab-induced cytokine production (Figure 9A) and cytolytic component production (Figure 9B) was assessed was used as a benchmark for safety assessment.

[0459] The #7 TriEngager-induced secretion level is significantly lower than Tarlatamab (Figure 9A). #7 TriEngager-induced cytolytic components production (Figure 9B) is also lower than Tarlatamab. The TriEngager plus IL-15 combination treatment leads to massive cytokine and cytolytic components secretion from PBMCs (Figures 9A and 9B). These results suggest using IL-15 with medium to low binding affinity to its receptors to avoid cytokine storm and other side effects while maintaining long-term tumor suppressive effects.

[0460] #11 TriEngager induces comparable cytokine and cytolytic component secretion to #2 Tarlatamab (Figures 10A and 10B). Based on TDCC data, the #11 TriEngager + IL-15 combination therapy was expected to induce very high cytotoxicity. Figure 10 shows that the #11 TriEngager + IL-15 combination therapy causes PBMCs to secrete significantly higher levels of cytokines and cytolytic components compared to #2 Tarlatamab.

[0461] #12 QuadEngager produced higher levels of cytokines and cytolytic components compared to #2 Tarlatamab and #11 TriEngager, indicating that #12 QuadEngager can induce stronger T cell activation (Figures 10A and 10B). However, unlike the #11 TriEngager + IL-15 combination therapy treatment group, the cytokines and cytolytic components produced by #12 QuadEngager did not show an “outburst effect” ( / .e. a rapid and uncontrolled release of cytokines, notably IFN-y, IL-6, TNF-a, and IL-10, from activated immune cells, which is a hallmark of cytokine release syndrome (CRS)). CRS is a significant safety concern in T-cell engager (TCE) therapies because it can cause severe inflammation, multi-organ failure, and even death if not managed properly. The lack of this outburst effect observed for #12 QuadEngager is advantageous because new immune cell engagers need to balance efficacy (tumor cell killing) with safety by reducing excessive cytokine release.

[0462] The results suggest that the function of IL-15 may be constrained when fused in #12 QuadEngager targeting tumor cells. Moreover, the advantage of #12 QuadEngager compared to #11 T riEngager plus IL-15 combination therapy lies in the fact that soluble cytokines act on every immune cell that has the corresponding cytokine receptor, without being restricted to target cells with a specific antigen.

[0463] Since Sushi-IL-15 is an IL-15 superagonist with stronger immune cell activation ability compared to parental IL-15, it was observed that the #13 QuadEngager induces higher secretion of cytokines and cytolytic components compared to both the #11 TriEngager plus IL-15 combination therapy group and the #12 QuadEngager at a higher treatment dose (370 pM) (Figures 10A and 10B).

[0464] 2.7 Engagers using VHH domains

[0465] HPN328 was used as a benchmark. T-cell dependent cellular cytotoxicity (TDCC) was tested using an Effector:Target ratio of 1 :1 (Effector: PBMCs, Target: luciferase SHP-77). HPN328 was shown to effectively induce TDCC (Figure 11).

[0466] #38 TriEngager was found to induce lower TDCC compared to #15 HPN328. This data shows that not all arbitrary combinations of binders into different structural format are functional. The combination therapy of #38 TriEngager and IL-15 induced TDCC well. #39 QuadEngager also induces poor TDCC effect compared to #15 HPN328 and other groups. It was hypothesized that the distance between IL-15 and the Fc fusion is too short, leading to a steric effect where the C-terminal IL-15 is positioned too close to the anti-CD3 arm. This proximity may restrict the spatial freedom of these binders, preventing them from adequately binding to their respective ligands or receptors, ultimately reducing T cell activation efficiency.

[0467] #40 QuadEngager induces better TDCC effect compared to #39 QuadEngager and other groups (Figure 11). It was hypothesized that the distance between IL-15 and the Fc fusion of the #40 QuadEngager is longer than #39 QuadEngager, therefore increase the flexibility of IL-15 and / or anti-CD3 binders and promote proper binding to their corresponding ligand / receptor. Therefore, the #40 QuadEngager induces better T cell activation and TDCC activity.

[0468] Improving the purity and stability of the #40 QuadEngager protein could further enhance TDCC.

[0469] 2.8 Conclusions

[0470] QuadEngagers for the treatment of SCLC were developed. These constructs incorporate anti-DLL3 binders for antigen-directed cell targeting, anti-CD3 binders for T cell activation, and IL-15-based cytokines, such as IL-15, sushi-IL-15 or IL-15-sushi, to enhance and sustain immune cell activation, proliferation, and reduce immune exhaustion (see Saito et al., Shock. (2020) 53(2):228-235 and Cai et al., Front Pharmacol. (2023) 12;14:1184703). Additionally, the inclusion of a human knob-into-holes IgG Fc domain extends half-life of the drug. Notably, IL-15, sushi-IL-15, or IL-15-sushi activate both T cells and natural killer (NK) cells, prevent immune cell exhaustion and sustaining a robust immune response.

[0471] This same concept can be adapted using the QuadEngager structural backbone by modifying its components, such as the tumor antigen-binding moiety, immune cell-binding moiety, cytokines, or other stimulatory elements like surface molecules and CD markers to sustained immune activation and prevent immune cell exhaustion (see e.g. see McLane et al., Annu Rev Immunol. (2019) 37:457-495, Barber et al., Nature. (2006) 439(7077):682-7, West et al., J Clin Invest. (2013) 123(6):2604-15, Lee et al., Front Immunol. (2023) 14:1117092 and Guo et al., Nat Immunol. (2021) 22(6):746-756, which are all hereby incorporated by reference in their entirety).

[0472] In conclusion, the fusion molecules developed herein offer a versatile and comprehensive approach to overcoming the limitations of current TCEs, providing the potential to improve therapeutic outcomes in cancer treatment.

[0473] Example 3: Materials and methods for Examples 4 to 6

[0474] 3.1 . T-cell dependent cellular cytotoxicity assay (normal conditions)

[0475] This assay investigates the ability of exemplary DLL3 targeting QuadEngager proteins to direct T cell mediated killing of a DLL3 expressing cancer cell line (SHP77) in normal conditions. The DLL3- expressing cell used in this study was engineered to express luciferase. For the TDCC assay (T cell dependent cellular cytotoxicity assay) T cells from one healthy donor (donor 1) and the DLL3-expressing cells were mixed and varying amounts of exemplary DLL3 targeting QuadEngager proteins were added to the mixture. The mixture was incubated for 48 hours at 37° C. As a positive control, parallel experiments were performed using a control bi-specific molecule targeting DLL3. After 48 hours, the remaining viable DLL3-expressing cells were quantified using a luminescence assay.

[0476] 3.2. T Cell Mediated Killing of a DLL3 Expressing Cancer Cell Line in Challenge Conditions

[0477] The aim of this study was to assess if exemplary DLL3 targeting QuadEngager molecules were able to direct T cells to kill a DLL3-expressing cell lines SHP77 in low E:T ratio. The DLL3-expressing cell used in this study was engineered to express luciferase.

[0478] For the TDCC assay (T cell dependent cellular cytotoxicity assay) T cells from one healthy donor (donor 1) and the DLL3-expressing cells were mixed and varying amounts of exemplary DLL3 targeting QuadEngager proteins was added to the mixture. The mixture was incubated for 48 hours at 37° C. As a positive control, parallel experiments were performed using a control bi-specific molecule targeting DLL3. After 48 hours, the remaining viable DLL3-expressing cells were quantified using a luminescence assay.

[0479] 3.3. T Cell Mediated Killing of a DLL3 Expressing Cancer Cell Line in Multi-Pay Repeated Tumor Cell Stimulation Assay

[0480] The aim of the study was to evaluate whether exemplary DLL3 targeting QuadEngager molecules could induce T-cell dependent killing of target cells - DLL3-expressing SHP-77 cells, sustain a durable immune response, and support T-cell fitness and persistence upon repeated tumor cell stimulation. SHP-77 cells expressing DLL3 were engineered to express luciferase. This was a multi-day time course study with assessments performed at regular 3-day intervals.

[0481] For the TDCC assay, Pan-T cells were isolated from PBMCs of one healthy donor and co-cultured with DLL3-expressing SHP-77 tumor cells at a 1 :1 effector-to-target (E:T) ratio. 5 nM exemplary DLL3- targeting QuadEngager proteins were added to the mixture on day 0. The cultures were maintained in a 37-degree incubator. At each time point (or every 3 days), an equal portion of the culture was sampled to assess TDCC activity by quantifying viable DLL3-expressing cells using a luminescence assay. Tumor- experienced T cells from the remaining mixture were isolated via a Ficoll density gradient and re-seeded with fresh tumor cells at a 1 :1 E:T ratio in the presence of 5nM QuadEngager proteins. As a positive control, parallel experiments were performed using a control bi-specific molecule targeting DLL3.

[0482] At multiple time points, parallel flow cytometry analyses were performed to assess T-cell function and phenotype and correlate these findings with potency. The assay concluded when T cells in the benchmark-treated group lost potency or when no viable cells remained. 3.4. PBMC Mediated Killing of a DLL3 Expressing Cancer Cell Line in Multi-Pay Repeated Tumor Cell Challenge Assay.

[0483] The aim of the study was to evaluate whether exemplary DLL3 targeting QuadEngager molecules could induce T-cell dependent killing of target cells - DLL3-expressing SHP-77 cells, sustain a durable immune response, and support T-cell fitness and persistence upon repeated tumor cell challenge. SHP-77 cells expressing DLL3 were engineered to express luciferase. This was a multi-day time course study with assessments performed at regular intervals.

[0484] For the PDCC (Peripheral blood mononuclear cell-dependent cellular cytotoxicity) assay, PBMCs from one healthy donor (donor 1) were cocultured with SHP-77 tumor cells expressing DLL3 at an effector-to- target (E:T) ratio of 0.25:1 or 1 :1 . Serially diluted QE proteins added at the start of incubation. As a positive control, parallel experiments were performed using a control bi-specific molecule targeting DLL3.

[0485] The PBMCs, tumor cells, and QuadEngager protein cultures were prepared in replicates, each corresponding to a specific time point, with additional replicates for parallel flow cytometry analysis. The cultures were maintained in a 37 degree incubator. At each time point, one replicate was used to assess the TDCC activity, in which the remaining viable DLL3-expressing cells were guantified using a luminescence assay. For the remaining replicates, an egual number of fresh tumor cells, eguivalent to the original input, were introduced to challenge the PBMCs.

[0486] The culture medium and QuadEngager proteins and positive control protein were refreshed every 6 days. At multiple time points, parallel flow cytometry analyses were performed to assess T-cell function and phenotype and correlate these findings with potency. The assay concluded when PMBCs in the benchmark-treated group lost potency or when no viable cells remained.

[0487] Example 4: Developing additional QuadEngager molecules

[0488] In this work, various multifunctional proteins (QuadEngager constructs) were designed, leveraging their multi-faceted immune cell activation to enhance tumor treatment effectiveness.

[0489] 4.1 Expression of Engagers and Combinations used in this study

[0490] Exemplary engagers were designed, expressed and purified as set out in Table 2.

[0491] Table 2: Engagers and Combinations used in this study

[0492] Example 5: Investigating additional QuadEnqaqer formats

[0493] Exemplary “P-shape” format molecules according to Figure 1 H were developed. These molecules investigate the use of Sushi-IL15 as the cytokine.

[0494] Such P-shape QuadEngagers show better T-cell mediated killing of a DLL3 expressing cancer cell line under challenge conditions than tarlatamab (using the method of Example 3.2). These formats also showed potential in terms of developability and potency (Table 3, Table 4, Figure 12). Table 3: Developability and potency of QuadEngager formats

[0495] Table 4: Efficacy of QuadEngager formats

[0496] This data demonstrates the promising therapeutic potential of the QuadEngagers, particular of the “P- shape” format. Exemplary “K-shape” format molecules according to Figure 1 K and 1 L were developed. These molecules investigate the use of Sushi-IL15 as the cytokine. The inventors further investigated the impact of different linker lengths between the domains of the “K-shape” QuadEngagers, as shown in Table 5. In particular, the inventors explored shortening the linker between the Fc region (CH3) and CD3 scFv.

[0497] The developability and potency of these molecules is shown in Table 5.

[0498] Table 5: Developability and potency of “K-shape” QuadEngager “walk-around” formats

[0499] The K-shape molecules were tested in an assay to investigate T-cell mediated killing of a DLL3 expressing cancer cell line under challenge conditions (according to Example 3.2). The results of the assay are shown in Figure 13A and 13B and Table 6. The developability and potency of these “K-shape” molecules was also investigated, as shown in Table 5.

[0500] As shown in Figure 13, the developed K-shape QuadEngagers demonstrated similar or improved cytotoxicity compared to control in a TDCC assay. Based on these results, the inventors hypothesized that these molecules may show improved cytotoxicity in restimulation assays, where T-cells are exhausted.

[0501] The “K-shape” walk around molecules demonstrated improved TDCC activity compared to control antibody, as well as good developability. Table 6: Efficacy of “K-shape” QuadEngager formats

[0502] Additional “K-shape” QuadEngager formats were investigated, using the method of Example 3.2. These “K-shape” QuadEngager formats demonstrated similar Max cytotoxic activity compared to Tarlatamab, and improved EC50 values, as shown in Table 7.

[0503] Table 7: Cytotoxicity of “K-shape” QuadEngager molecules

[0504] Example 6: Efficacy of QuadEnqaqers in rechallenqe assays

[0505] The P-shape QuadEngager molecules were tested for their ability to direct PBMC mediated killing of a DLL3 expressing cancer cell line in a multi-day repeated tumor cell challenge assay (as described in Example 3.4). The results are shown in Figure 14.

[0506] As shown, these molecules demonstrate improved cytotoxicity compared to control antibody, both in terms of EC50, and in terms of max killing achieved.

[0507] Exemplary “K-shape” molecules were also tested in challenge assays (according to the methods of Example 3.3. and 3.4.), alongside exemplary “P-shape” molecules. The results of these assays are shown in Figure 15A and 15B respectively. This data demonstrates the improvements in EC50 and / or max killing demonstrated by the QuadEngager formats compared to control antibody, particularly under challenge conditions.

[0508] Conclusions

[0509] QuadEngagers for the treatment of SCLC were developed. The program hypothesis is that in aggressive solid tumors — either before treatment or after tarlatamab — suboptimal response and durability are driven by exhausted immune cells, primarily T cells. These constructs incorporate tumor antigen binders for precise tumor targeting, immune-cell binders for T cell activation, and IL-15-based cytokines, including IL-15 / Sushi domain fusion proteins. Notably, IL-15, sushi-IL-15, or IL-15-sushi activate both T cells and natural killer (NK) cells, combating immune cell exhaustion and sustaining a robust immune response.

[0510] Exemplary QuadEngager molecules were investigated in TDCC assays, including under challenge conditions. Various QuadEngager molecules demonstrated improved cytotoxicity compared to control antibody (either in terms of EC50, max killing, or both). Furthermore, these improvements in cytotoxicity were particular observed under re-challenge / restimulation conditions, indicating the utility of the QuadEngager molecules in conditions where T-cell exhaustion presents a therapeutic challenge (e.g. in cancer, e.g. in solid tumors).

[0511] In conclusion, the fusion molecules developed herein offer a versatile and comprehensive approach to overcoming the limitations of current TCEs, providing the potential to improve therapeutic outcomes in cancer treatment.

[0512] Example 7: Additional construct development using different DLL3 binders

[0513] TriEngager and QuadEngager constructs were developed with alternative DLL3 binders incorporated. The developability of these molecules is shown in Table 8 and 9. Several of the tested constructs comprising different DLL3 binders demonstrated similar TDCC, indicating that the efficacy of the QuadEngager formats is not limited to specific binder sequences.

[0514] Table 8: Developability of additional constructs

[0515] Table 9: Developability of additional constructs

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Claims

Claims:1 . A fusion molecule comprising an immune cell engager and a cytokine.

2. The fusion molecule of claim 1 , wherein the immune cell engager comprises an immune cellbinding moiety, a tumor antigen-binding moiety and an Fc region.

3. The fusion molecule of claim 1 or claim 2, wherein the fusion molecule is a polypeptide complex comprising, or consisting of:(i) a first polypeptide comprising a tumor antigen-binding moiety, an immune cell-binding moiety, and a first CH2-CH3 region; and(ii) a second polypeptide comprising a second CH2-CH3 region and a cytokine.

4. The fusion molecule of claim 3, wherein the fusion molecule is a polypeptide complex comprising, or consisting of:(i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, an immune cell-binding moiety, and a first CH2-CH3 region; and(ii) a second polypeptide comprising from N to C terminus: a second CH2-CH3 region and a cytokine.

5. The fusion molecule of claim 3, wherein the fusion molecule is a polypeptide complex comprising, or consisting of:(i) a first polypeptide comprising from N to C terminus: a tumor antigen-binding moiety, a first CH2-CH3 region, and an immune cell-binding moiety; and(ii) a second polypeptide comprising from N to C terminus: a second CH2-CH3 region and a cytokine.

6. The fusion molecule of claim 3 to claim 5, wherein the second polypeptide comprises a linker between the second CH2-CH3 region and the cytokine, optionally wherein the linker comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 29, 31 , 32, 68, 69, 70, 71 , 72, 73, 90 or 91 .

7. The fusion molecule according to any one of claims 1 to 6, wherein the Fc region comprises at least one modification to promote heterodimerization, prevent antibody-dependent cellular cytotoxicity and / or extend serum half-life.

8. The fusion molecule according to any one of claims 1 to 7, wherein the Fc region comprises knob-in-hole modifications.

9. The fusion molecule according to any one of claims 1 to 8, wherein the cytokine is IL-15, IL-2, IL- 4. IL-7, IL-9, IL-10, IL-12, IL-18, IL-21 , or is an engineered cytokine.

10. The fusion molecule according to any one of claims 1 to 9, wherein the cytokine is an engineered cytokine.

11. The fusion molecule according to any one of claims 1 to 10, wherein the cytokine is sushi-IL-15, IL-15-sushi or Neo-2 / 15.

12. The fusion molecule according to any one of claims 1 to 8, wherein the cytokine is IL-15, or an IL- 15 / sushi domain fusion construct.

13. The fusion molecule according to any one of claims 1 to 12, wherein the immune cell-binding moiety is a T-cell binding moiety.

14. The fusion molecule according to any one of claims 1 to 13, wherein the immune cell-binding moiety is a scFv, VH domain, Fab or nanobody.

15. The fusion molecule according to any one of claims 1 to 14, wherein the immune cell-binding moiety specifically binds CD2, CD3, CD16, CD28, CD54, CD137 or CD335.

16. The fusion molecule according to any one of claims 1 to 15, wherein the immune cell-binding moiety specifically binds CD3.

17. The fusion molecule according to any one of claims 1 to 16, wherein the tumor antigen-binding moiety is a scFv, VH domain, Fab or nanobody.

18. The fusion molecule according to any one of claims 1 to 17, wherein the tumor antigen-binding moiety specifically binds a cancer cell antigen or a cancer-associated antigen.

19. The fusion molecule according to any one of claims 1 to 18, wherein the tumor antigen-binding moiety specifically binds an antigen selected from DLL3, EpCAM, TROP2, SEZ6, KLK2, PSMA, STEAP1 , STEAP2, CLDN6 and MUC16.

20. The fusion molecule according to any one of claims 1 to 19, wherein the tumor antigen-binding moiety specifically binds DLL3.21 . A polynucleotide or plurality of polynucleotides encoding the fusion molecule according to any one of claims 1 to 20.

22. A pharmaceutical composition comprising a fusion molecule according to any one of claims 1 to 20, or a polynucleotide according to claim 21 , and a pharmaceutically acceptable carrier.8323. A fusion molecule according to any one of claims 1 to 20, a polynucleotide according to claim 21 , or a pharmaceutical composition according to claim 22, for use in a method of treating or preventing a disease or condition.

24. A method of treating or preventing a disease or condition, comprising administering a fusion molecule according to any one of claims 1 to 20, a polynucleotide according to claim 21 , or a pharmaceutical composition according to claim 22, to a patient in need thereof.

25. The fusion molecule for use according to claim 23 or the method according to claim 24, wherein the disease or condition is cancer.

26. The fusion molecule for use or the method according to claim 25, wherein the cancer is small cell lung cancer, non-small cell lung cancer, neuroendocrine cancer, prostate cancer, breast cancer, gastric cancer, pancreatic cancer, liver cancer, lymphoma, leukaemia, myeloma, brain tumors, skin cancer, uterine cancer, and kidney cancer; optionally wherein the neuroendocrine cancer is NEPC, LCNEC or NEBC.

27. A method of treating cancer, comprising administering a fusion molecule comprising an immune cell-binding moiety, a tumor antigen-binding moiety, an Fc region and a cytokine.

28. A fusion molecule comprising an immune cell-binding moiety, a tumor antigen-binding moiety, an Fc region and a cytokine for use in a method of treating cancer.

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