Tumor necrosis factor superfamily ligand-based multifunctional protein platform

Abstract

The present application discloses multifunctional and / or multispecific proteins that include at least one functional moiety. Ine one embodiment, a multifunctional molecule comprises a trimeric ligand with three monomer molecules, wherein each monomer molecule is selected from a tumor necrosis factor superfamily (TNFSF) member, a TNFSF-like member, or a combination thereof. At least one of the monomer molecules comprises a modification at one or more amino acid positions that forms a new covalent and / or non-covalent association between at least two of the monomers, and at least one functional moiety is fused to either the N-terminus or C-terminus of one of the monomer molecules. The multifunctional and / or multispecific proteins can be used in diagnostic and purification assays as well as therapeutic methods of treatment.

Description

Docket No. BREACH-001 / WOOlTUMOR NECROSIS FACTOR SUPERFAMILY LIGAND-BASED MULTIFUNCTIONAL PROTEIN PLATFORM PRIORITY CLAIMThis application claims priority to U.S. Provisional Patent Application No. 63 / 724,160, filed on November 22, 2024, and U.S. Provisional Patent Application No. 63 / 752,044, filed on January 31, 2025, the contents of each of which are hereby incorporated by reference in their entirety.SEQUENCE LISTINGThe present application is accompanied by a sequence listing submitted as an electronically filed XML document entitled “2025-11-16 BREACH-001 -WOO 1 Sequence Listing.xml”, created on November 16, 2025, and having a size of 34.4 KB. The content of this sequence listing is hereby incorporated by reference in its entirety.BACKGROUNDThe tumor necrosis factor superfamilies of ligands (TNFSF) and receptors (TNFRSF) provide key communication signals between various cell types during development, especially in the skin, bones, and lymphoid organs, and maintain organ homeostasis and initiate tissue responses. TNFSFs are essential for innate and adaptive immunity and are implicated in diverse pathological conditions such as autoimmune diseases, developmental abnormalities, and cancer (Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell (2001) 104(4):487-501; Dostert C, GrusdatM, Letellier E, Brenner D. The TNF Family of Ligands and Receptors: Communication Modules in the Immune System and Beyond. Physiol Rev (2019) 99(1): 115-60). TNFSFs activate signaling pathways that mediate pro-tumor effects like inflammation and cell survival, and anti-tumor processes like apoptosis (Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol (2003) 3(9):745- 56.). The TNF-related ligands are defined by structural homology in their ectodomain and assemble into trimers that form a highly efficient receptor-clustering and signal-initiating mechanism (Bodmer et al., 2002). TNF receptors (TNFRs) share a conserved ectodomain defined by a cysteine-rich signature. High-affinity binding of their specific TNFSF ligands induces clustering of receptors expressed in the cognate target cell, which in turn initiates signal-transduction pathways culminating in cellular responses. The cytosolic signaling domain subdivides TNFRSF receptors into those utilizing the death domain, those engaging the TRAF family of ubiquitin E3 ligases (Li et al., 2013), and those lacking a cytosolic domain and functioning as decoy receptors. Depending upon the specific cellular circumstance, the outcome of TNFR signaling can be cellular life, death, or differentiation. ADocket No. BREACH-001 / WOOl subset of TNF superfamily members provides co-stimulatory or co-inhibitory signals essential for innate and adaptive immunity with an emphasis on T cell responses. The TNFRs with a co-stimulatory reputation are encoded by genes residing within an immune-response locus in chromosomal region lp36 and include GITR (glucocorticoid-induced tumor necrosis factor), 0X40, HVEM (herpesvirus entry mediator), DR3 (death receptor 3), 4-1BB, CD30, and TNFR2 (tumor necrosis factor receptor 2); these are derived from genomic evolution in chromosomal region 12pl3, encoding CD27, LTbetaR, and TNFR1. Their corresponding TNFSF ligands are encoded by genes residing within the major histocompatibility complex (MHC) paralogous regions on chromosomes 1, 6, 9, and 19. However, the extensive shared ligand and receptor usage of TNFSF ligands by several of the receptors creates a communication network among distinct cells and tissues that regulate co-stimulatory and inhibitory pathways, providing mechanisms for initiating immunity and resetting homeostasis.BRIEF SUMMARYThe present disclosure provides compositions and methods related to polarized trimeric TNFSF ligand fusion proteins and multifunctional / multispecific proteins built upon polarized trimeric TNFSF ligand fusion proteins. The inventor has recognized that the biologic activity of TNFSF ligands and their unique homotrimeric architecture can be leveraged to create new multifunctional proteins with unique properties and uses. As described in more detail herein, structural and protein engineering approaches can be used to modify TNFSF ligands to create trimers (e.g., polarized trimers) that can be functionalized for use as multi-specific and multifunctional fusion proteins. This is particularly useful in the context of multifunctional immune cell engagers, such as T cell and NK cell engagers, since some TNFSF ligands such as LIGHT, TL1A, 4-1BBL, and CD40L are T / NK cell co-stimulators. The multifunctional proteins built upon these T / NK cell costimulatory TNFSF ligands represent a new family of T / NK cell engager s / co- stimulators with unparalleled multifunctional potential.In some embodiments, the present disclosure provides polarized trimeric TNFSF ligands which serve as a central connector to create multifunctional / multispecific proteins such as T cell engagers / co-stimulators. The disclosure provides mutations of TNFSF ligands as well as multiple potential architectures built upon the polarized TNFSF trimer. In particular TNFSF ligands that exhibit immune cell activation such as LIGHT, TL1A, 4-1BBL, CD40L (among others), can be modified to form polarized trimers that can be fused with up to six different fusion partners at the first degree of interaction. The fusion partners can be binders (scFv, Fv, Fab, single domain antibodies, peptides, etc.) to immune cell stimulating antigens such as CD3 and CD16, tumor associated antigens,Docket No. BREACH-001 / WOOl checkpoints, and a half-life extension such as an Fc, a anti-human serum albumin binder, etc. The number of potential fusion partners enables the design of complex multispecific fusion proteins that can address different targets and pathways in one single molecule. In addition, the TNFSF mutations by virtue of stabilizing interaction between specific monomers lead to increased thermostability of the overall trimer, making these proteins better candidates than their wild type counterparts for therapeutic development. Furthermore, systemic cytokine therapy is generally limited by rapid degradation and elimination of the cytokine, the inability to achieve optimal concentrations in the tumor, and dosedependent toxicity, including life-threatening side effects such as vascular leak syndrome and orthostatic hypotension. As fusion proteins combining TAA, HLE, and other targeting moieties, the polarized TNFSF ligand-multispecific fusion proteins are expected to limit or abrogate these limitations.In one embodiment, a multifunctional molecule comprises a trimeric ligand with three monomer molecules. Each monomer molecule is selected from a tumor necrosis factor superfamily (TNFSF) member, a TNFSF-like member, or a combination thereof. At least one monomer molecule comprises a modification at one or more amino acid positions that forms one or more new covalent and / or non-covalent associations between at least two of the monomers, and at least one functional moiety is fused to either the N-terminus or C-terminus of one of the monomer molecules.In some embodiments, the trimeric ligand comprises a first pair of complementary amino acid modifications, the one or more pairs comprising a first pair comprising an amino acid modification in a first monomer molecule and a corresponding amino acid modification in a second monomer molecule, wherein the amino acid modifications form a new covalent and / or non-covalent association between the monomers. In some embodiments, the trimeric ligand comprises a second pair of complementary amino acid modifications, wherein the second pair comprises a second amino acid modification in the first monomer molecule and a corresponding amino acid modification in a third monomer molecule, wherein the amino acid modifications form a new covalent and / or non-covalent associations between the monomers. In some embodiments, the trimeric ligand comprises one or more pairs of complementary amino acid modifications, each pair forming a new covalent and / or non- covalent association between the monomers. In some embodiments, the new covalent or non-covalent associations cause the three monomer molecules to associate in a specific configuration to create the trimeric ligand.In some embodiments, a pair of complementary amino acid modifications comprises a modification at an amino acid position in a first monomer and a modification at an amino acid positionDocket No. BREACH-001 / WOOl in a second monomer, wherein the atoms in the amino acid positions in the first and second monomers that engage in interchain interaction within the trimeric ligand are between 2-6 angstroms from one another.In some embodiments, the covalent associations comprise disulfide bonds created by new cysteine residues introduced into at least two monomers. In some embodiments, the non-covalent associations comprise salt bridges, hydrogen bonds, and / or hydrophobic interactions created by new residues introduced into the at least two monomers.In any embodiment, the trimeric ligand retains its ability to bind to its cognate receptors.In some embodiments, the monomer molecules are TNFSF members, and are selected from the group consisting of: TNF-alpha, LT-alpha, LT-beta, OX40L, CD40L, FasL, CD27L, CD30L, 4- 1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, TL1A, GITRL, EDA-A1, and EDA-A2.In some embodiments, the monomer molecules comprise LIGHT, and the modifications that form a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: N93, R124, S182, Q183, S185, G188, R189, T191, R195, S200, R232, and V240, as compared to wild-type LIGHT. In some embodiments, one of the monomer molecules further comprises an additional C-terminal extension sequence of R241X242X243D244, X241X242X243X244, or X241X242X243, wherein X242 or X243 comprise a cysteine residue. In some embodiments, the modifications that form a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: N93C, R124A, R124L, S182C, S182D, S182F, S182H, S182K, S182R, S182W, Q183R, S185E, G188C, T191C, R195D, S200A, S200C, S200D, S200F, S200H, S200K, S200W, R232D, V240D, X242C, and X243C, as compared to wild-type LIGHT. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: 93 / 242; 93 / 243; 182 / 200; 183 / 232; 188 / 191; 189 / 185; 185 / 195; and 124 / 240, as compared to wildtype LIGHT. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: N93C / 242C; N93C / 243C; S182C / S200C; G188C / T191C; R124 / V240D; S182R / S200D; S182K / S200D; S182F / S200F; S182H / S200H; Q183R / R232D; R189D / R195; and S185E / R195, as compared to wild-type LIGHT. In some embodiments, one, two, or three of the monomer molecules further comprise one or more amino acid modifications selected from the groupDocket No. BREACH-001 / WOOl consisting of: R124A, R124L, Y142H, Y144H, S182A, S185E, R189D, R195D, S200A, F202Y, or F238Y, as compared to wild-type LIGHT.In some embodiments, the monomer molecules comprise CD27L, and the modification that forms a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of SI 17 and Q149, as compared to wild-type CD27L. In some embodiments, the modification that forms a new covalent or non- covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: Q149C and SI 17C, as compared to wild-type CD27L. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following paired amino acid modifications in at least two monomers: 117 / 149, as compared to wild-type CD40L. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: Q149C / S117C, as compared to wild-type CD27L. In some embodiments, one, two, or three of the monomer molecules further comprise one or more amino acid modifications selected from the group consisting of: S117C, SI 17W, Q149C, and Q149Y as compared to wild-type CD27L. In such embodiments, these mutations can lead to the covalent interaction between at least two monomers, thereby promoting a polarized trimeric arrangement.In some embodiments, the monomer molecules comprise CD40L, and the modification that forms a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of A208, T211, S213, S222, Cl 78, C218, and G219, as compared to wild-type CD40L. In some embodiments, the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: C178S, A208C, A208D, T211C, S213C, C218S, G219C, S222R, S222C, and S222K, as compared to wildtype CD40L. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following paired amino acid modifications in at least two monomers: 178 / 218; 208 / 222; 211 / 213 and 211 / 219, as compared to wild-type CD40L. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: A208D / S222R; A208C / S222C; A208D / S222K; and T211C / S213C; T211C / G219C, as compared to wild-type CD40L.Docket No. BREACH-001 / WOOlIn some embodiments, the monomer molecules comprise TL1A, and the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of R96, D146, M197, and D242, as compared to wild-type TL1A. In some embodiments, the modifications that form a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: R96D, D146R, D146K, D146N, M197C, and D242C as compared to wild-type TL1 A. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following paired amino acid positions in at least two monomers: 197 / 242; and 146 / 96, as compared to wild-type TL1A. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: M197C / D242C; R96D / D146R; and M197C / D242C, as compared to wild-type TL1A. In some embodiments, one, two, or three of the monomer molecules further comprise one or more amino acid modifications selected from the group consisting of: C163S, C203S, F148Y, and F244Y, as compared to wild-type TL1 A.In some embodiments, the monomer molecules comprise LTB, and the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: L138, G181, and S204, as compared to wild-type LTB. In some embodiments, the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: L138K, L138R, G181C, G181D, and S204C, as compared to wild-type LTB. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following paired amino acid positions in at least two monomers: 181 / 204; and 138 / 181, as compared to wild-type LTB. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: L138R / G181D; L138K / G181D; and G181C / S204C, as compared to wild-type LTB.In some embodiments, the monomer molecules comprise LTA / LTB / LTB heterotrimer, and the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: (LTA)Ml 54, (LTA)L164, (LTB)R160, (LTB)A192, (LTB)G191, and (LTB)Y212 as compared to wild-type LTA and LTB. In some embodiments, the modification that forms a new covalent or non-covalentDocket No. BREACH-001 / WOOl association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: (LTA)M154C, (LTA)L164E, (LTB)R160E, (LTB)A192C, (LTB)G191C, and (LTB)Y212C, as compared to wild-type LTA and LTB. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following paired amino acid positions in at least two monomers: (LTA)164 / (LTB)160; (LTB)192 / (LTB)212 and (LTA)154 / (LTB)212, as compared to wild-type LTA and LTB. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: (LTA)L164E / (LTB)R160; (LTB)A192C / (LTB)Y212C; (LTB)G191C / (LTB)S214C; and(LTA)M154V / (LTB)Y212C, as compared to wild-type LTA and LTB.In some embodiments, the monomer molecules comprise 4-1BBL, and the modification that forms a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of G80, D184, R193 and T241, as compared to wild-type 4-1 BBL. In some embodiments, the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: G80C, D184C, R193C, and T241C as compared to wild-type 4-1BBL. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following paired amino acid modifications in at least two monomers: 184 / 193; and 80 / 241, as compared to wild-type 4-1BBL. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: D184C / R193C and G80C / T241C, as compared to wild-type 4-1BBL.In some embodiments, the monomer molecules comprise TRAIL, and the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: D203, C230, and S232, as compared to wild-type TRAIL. In some embodiments, the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: D203C, C230H, and S232C, as compared to wildtype TRAIL. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following paired amino acid positions in at least two monomers: 230 / 230; and 203 / 232, as compared to wild-type TRAIL. In some embodiments, the new covalent and / or non-covalent association formed between the monomers comprises one or moreDocket No. BREACH-001 / WOOl of the following pairs of amino acid positions in at least two monomers: D203C / S232C, as compared to wild-type TRAIL.In some embodiments, the trimeric ligand further comprises a modification at one or more amino acid positions to remove unpaired cysteine residues.In some embodiments, the trimeric ligand comprises two, three, four, five, or six functional moieties, wherein each functional moiety is fused to either the N-terminus or C-terminus of one of the monomer molecules. In some embodiments, the functional moieties comprise: (i) a tumor-targeting moiety that binds to a cancer antigen; and one, two, three, four, or five of: (ii) an immune cell engager; (iii) a cytokine molecule; (iv) a stromal modifying moiety; (v) an immune checkpoint inhibitor; (vi) an enzyme; and (vii) a half-life extension moiety. In some embodiments, if (ii) and (iii) are absent, then (i) and (iv) are present; if one (i) and one (ii) are present, then (iii) or (iv) or both are present; and if one (i) and one (iii) are present, then (ii) or (iv) or both are present.In some embodiments, the trimeric ligand comprises at least one tumor-targeting moiety that binds to a cancer antigen. In some embodiments, the trimeric ligand comprises at least two tumor targeting moieties that bind to a cancer antigen. In some embodiments, the trimeric ligand comprises at least three tumor targeting moieties that bind to a cancer antigen. In some embodiments, the tumor targeting moieties bind to different cancer antigens. In some embodiments, the tumor-targeting moiety localizes the trimeric ligand to the cancer, thereby activating the immune system against the cancer.In some embodiments, the cancer antigen is selected from the group consisting of: Delta-like protein 3 (DLL3), B-cell maturation antigen (BCMA), carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), epithelial cell adhesion molecule (EpCAM), CD20, CD26, CD123, CD30, CD33, CD47, CD52, CD133, glycoprotein A33 (gpA33), mucins, tumor associated glycoprotein-72 (TAG-72), type IX collagen (CIX), glutamate carboxypeptidase II (PSMA), folate-binding protein, GD2, GD3, GM2, vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), integrin, aVp3, a501, ERBB2, ERBB3, mesenchymal epithelial transition (MET), insulin-like growth factor-I receptor (IGFIR), ephrin type-A receptor 3 (EPHA3), TRAIL receptor 1 (TRAILR1), TRAIL receptor 2 (TRAILR2), receptor activator of nuclear factor kappa beta (RANKL), fibroblast activation protein (FAP), claudin 18.2, mesothelin, receptor tyrosine kinase like orphan receptor 1 (ROR1), epidermal growth factor receptor variant III (EGFRVIII), six-transmembrane epithelial antigen of the prostate- 1 (STEAP1) six -transmembrane epithelial antigen of the prostate-2 (STEAP2), B7-H3 (CD276), orphanDocket No. BREACH-001 / WOOlG protein-coupled receptor, class C group 5 member D (GPRC5D), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), and tenascin.In some embodiments, the trimeric ligand comprises at least one immune cell engager. In some embodiments, the trimeric ligand comprises at least two immune cell engagers. In some embodiments, the immune cell engager comprises a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, a myeloid cell engager, and / or a Toll-Like Receptor (TLR).In some embodiments, the trimeric ligand comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of: PD-1, PD- Ll, and CTLA-4.In some embodiments, the trimeric ligand comprises a stromal modifying moiety. In some embodiments, the stromal modifying moiety is selected from the group consisting of: a vascular modifying moiety (VEGF), Fibroblast association protein (FAP), and extracellular matrix binding protein.In one embodiment, a multifunctional molecule comprises a first tumor targeting moiety targeting PD-L1, a second tumor targeting moiety comprising a tumor targeting peptide, and a halflife extension moiety. In some embodiments, the first tumor targeting moiety comprises an anti-PD- L1 VHH fused to at least one monomer. In such embodiments, the anti-PD-Ll VHH comprises a VHH sequence selected from one of SEQ ID Nos: 27-32. In some embodiments, the first tumor targeting moiety comprises an anti-PD-Ll Fv comprising a VH and a VL, wherein the VH and VL are each fused to at least one monomer. In some embodiments, the multifunctional molecule further comprises a third tumor targeting moiety comprising a VEGF targeting moiety.In one embodiment, a multifunctional molecule comprises a first tumor-targeting moiety targeting DLL3, a second tumor targeting moiety comprising a tumor targeting peptide, a half-life extension moiety, and an immune cell engager. In some embodiments, the first tumor-targeting moiety comprises an anti-DLL3 VHH fused to at least one monomer. In some embodiments, the first tumor-targeting moiety comprises an anti-DLL3 Fv comprising a VH and a VL, wherein the VH and VL are each fused to at least one monomer. In some embodiments, the immune cell engager targets CD3. In some embodiments, the immune cell engager comprises an anti-CD3 VHH.In some embodiments, the half-life extension moiety comprises a human serum albumin (HSA) binding protein. In some embodiments, the trimeric ligand is LIGHT.Docket No. BREACH-001 / WOOlIn some embodiments, the multifunctional molecule promotes T cells, Myeloid cells, Dendritic cells, or cytokine immunity against target cells, e.g., cancer.In one embodiment, a pharmaceutical composition comprises (a) a multifunctional molecule as disclosed herein, and (b) a pharmaceutically acceptable carrier. In one embodiment, one or more nucleic acids encode a multifunctional molecule as disclosed herein. In one embodiment, an expression vector comprises the one or more nucleic acids. In one embodiment, a host cell comprises the one or more nucleic acids or the expression vector. In one embodiment, a method of making a multifunctional molecule comprises culturing the host cell and recovering the multifunctional molecule from the cell culture.In one embodiment, a method of treating a disease or condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a multifunctional molecule as disclosed herein. In some embodiments, the disease or condition comprises a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus- host disease or a host-versus-graft disease.In one embodiment, a method of treating cancer in a patient comprises administering a composition comprising a multifunctional molecule as disclosed herein. In some embodiments, the cancer is selected from the group consisting of renal clear cell carcinoma (RCC), lung cancer, nonsmall cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, gastric adenocarcinoma, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer (TNBC), head and neck tumor, colorectal adenocarcinoma, melanoma, metastatic melanoma, gallbladder carcinoma, liver carcinoma, colon carcinoma, pancreatic carcinoma, bone sarcoma, thyroid adenocarcinoma, and renal cell carcinoma. In some embodiments, the patient experiences an increase in tumor growth inhibition of at least about 10%, 20%, 30% 40% 50% 60% 70% 80% 90%, 100%, 125%, 150%, 175%, 200% 225% 250%, 275%, 300%, 325% 350%, 375%, 400%, 425%, 450%, 475%, 500%, 525%, 550%, 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000%, as compared to a control or an untreated patient. In some embodiments, the patient experiences a decrease in tumor growth inhibition of at least about 10%, 20%, 30% 40% 50% 60% 70% 80% 90%, 100%, 125%, 150%, 175%, 200% 225% 250%, 275%, 300%, 325% 350%, 375%, 400%, 425%, 450%, 475%, 50Q%, 525%, 550%, 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000%, as compared to a control or an untreated patient.Docket No. BREACH-001 / WOOlIn one embodiment, a method of reducing a tumor comprising contacting the tumor with a composition comprising a multifunctional molecule as disclosed herein. In one embodiment, a method of reducing a tumor in a subject in need thereof comprising administering to the subject a composition comprising a multifunctional molecule as disclosed herein. In one embodiment, a method of treating a subject having a cancer comprises administering to the subject a composition comprising a multifunctional molecule as disclosed herein.In any embodiment, the subject can be a human subject.In one embodiment, variant LIGHT (TNFSF14) molecules are provided. In some embodiments, the variant LIGHT molecule comprises a modification at one or more amino acid positions selected from the group consisting of R124, Y142, Y144, S182, G188, R189, T191, R195, S200, F202, F238, and V240, as compared to wild-type LIGHT. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomers in the trimeric ligand that one or both positions can be modified to create a new covalent and / or non-covalent association, as further described herein: S182 / S200, G188 / T191, R124 / V240; R189 / R195; and S185 / R195. In some embodiments, if using R124 / V240 mutations to form a salt bridge between the two residues, R124 may be wild-type, whereas V240 may be modified to aspartic acid (V240D). In some embodiments, the variants aforementioned are mutations to residues that strengthen the interaction between the monomers. In some embodiments, the monomer molecules are selected from and paired according to Table 3.In one embodiment, variant CD27L (TNFSF7) molecules are provided. In some embodiments, the variant CD27L molecule comprises a modification at one or more amino acid positions selected from the group consisting of Q149 and SI 17. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both positions can be modified to create a new covalent or non-covalent association, as further described herein: S117 / Q149. In some embodiments, the variants aforementioned are mutations to cysteines. In some embodiments, the variants aforementioned are mutations to residues that strengthen the interaction between the monomers. In some embodiments, the monomer molecules are selected from and paired according to Table 4.In one embodiment, variant CD40L (TNFSF5) molecules are provided. In some embodiments, the variant CD40L molecule comprises a modification at one or more amino acid positions selected from the group consisting of A208, T211, S213, G219, and S222. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a secondDocket No. BREACH-001 / WOOl monomer in the trimeric ligand that one or both positions can be modified to create a new covalent or non-covalent association, as further described herein: A208 / S222, T211 / S213 and T211 / G219. In some embodiments, the variants aforementioned are mutations to cysteines. In some embodiments, the variants aforementioned are mutations to residues that strengthen the interaction between the monomers. In some embodiments, the monomer molecules are selected from and paired according to Table 5.In one embodiment, variant TL1A (TNFSF15) molecules are provided. In some embodiments, the variant TL1A molecule comprises a modification at one or more amino acid positions selected from the group consisting of R96, D146, F148, C163, M197, C203, D242, and F244. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both positions can be modified to create a new covalent or non-covalent association, as further described herein: M197 / D242 and R96 / D146. In some embodiments, the variants aforementioned are mutations to cysteines. In some embodiments, the variants aforementioned are mutations to residues that strengthen the interaction between the monomers. In some embodiments, the monomer molecules are selected from and paired according to Table 6.In one embodiment, variant LTB (TNFSF3) molecules are provided. In some embodiments, the variant LTB molecule comprises a modification at one or more amino acid positions selected from the group consisting of L138, G181, and S204. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both positions can be modified to create a new covalent or non-covalent association, as further described herein: G181 / S204 and L38 / G181. In some embodiments, the variants aforementioned are mutations to cysteines. In some embodiments, the variants aforementioned are mutations to residues that strengthen the interaction between the monomers. In some embodiments, the monomer molecules are paired according to Table 7.In one embodiment, variant LTA (TNFSF1) and LTB (TNFSF3) molecules are provided. In some embodiments, the variant LTA / LTB molecule comprises a modification at one or more amino acid positions selected from the group consisting of (LTA)M154, (LTA)L164, (LTB)R160, (LTB)A192, (LTB)G191, and (LTB)Y212. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both positions can be modified to create a new covalent or non-covalent association, as further described herein: (LTA)L164E / (LTB)R160; (LTB)A192C / (LTB)Y212C;Docket No. BREACH-001 / WOOl(LTB)G191C / (LTB)S214C; and (LTA)M154V / (LTB)Y212C. In some embodiments, the variants aforementioned are mutations to cysteines. In some embodiments, the variants aforementioned are mutations to residues that strengthen the interaction between the monomers. In some embodiments, the monomer molecules are selected from and paired according to Table 8.In one embodiment, variant 4-1BBL (TNFSF9) molecules are provided. In some embodiments, the variant 4-1BBL molecule comprises a modification at one or more amino acid positions selected from the group consisting of G80, DI 84, R193, and T241. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both positions can be modified to create a new covalent or non-covalent association, as further described herein: D184 / R193 and G80 / T241. In some embodiments, the variants aforementioned are mutations to cysteines. In some embodiments, the variants aforementioned are mutations to residues that strengthen the interaction between the monomers. In some embodiments, the monomer molecules are paired according to Table 9.In one embodiment, variant TRAIL (TNFSF10) molecules are provided. In some embodiments, the variant TRAIL molecule comprises a modification at one or more amino acid positions selected from the group consisting of D203, C230, and S232. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both positions can be modified to create a new covalent or non-covalent association, as further described herein: D203 / S232. In some embodiments, the variants aforementioned are mutations to cysteines. In some embodiments, the variants aforementioned are mutations to residues that strengthen the interaction between the monomers. In some embodiments, the monomer molecules are paired according to Table 10.INCORPORATION BY REFERENCEAll publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DISCLOSURE OF THE DRAWINGSThe novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained byDocket No. BREACH-001 / WOOl reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:FIG. 1A is a flowchart depicting a general strategy for creating polarized trimeric ligands comprising up to six functional moieties; and FIG. IB is a diagram depicting the creation of new covalent and / or non-covalent associations within a trimeric ligand.FIG. 2 is a diagram depicting the addition of certain mutations to monomers of a trimeric ligand to increase overall stability.FIGS. 3A-M are diagrams showing examples of multifunctional and / or multispecific formats that are enabled by a polarized TNFSF ligand structure, e.g.: the polarized TNFSF ligand can be fused to an Fc via the C-terminus of one or two polarized monomers; the polarized TNFSF ligand can be fused to 6 different partners (for example 6 different VHHs); the polarized TNFSF ligand can be fused to an scFv; the polarized TNFSF ligand can be fused to a Fv; the polarized TNFSF ligand can be fused to a cytokine; the polarized TNFSF ligand can be fused to a combination of different types of interactors including tumor associated antigen binders (TAAs), checkpoint inhibitors (CPI), CD3 binders (CD3), cleavable masks, half-life extension moieties (HLE). FIGS. 3N-P are diagrams showing examples of antibody, and antibody-derived proteins reformatting through polarized TNF trimer fusions according to the disclosure.FIGS. 4A-B depict SDS-PAGE gel analyses and chromatographs of two embodiments of trimeric ligands according to the disclosure, illustrating their ability to associate into oligomers.FIG. 5 depicts ELISA analyses of various trimeric ligands according to the disclosure, showing whether each trimeric ligand retains its innate ability to bind to certain receptors.FIG. 6 depicts a comparison between a functional VHH-fused LIGHT trimeric ligand and the same VHH fused to an Fc in their ability to bind to the VHH’s target protein.FIG. 7A depicts a three-dimensional view of a Trastuzumab heavy chain (HC) and light chain (LC) variable domains fused to a LIGHT monomer at its N- and C-termini; FIG. 7B is a graph comparing the ability of the Trastuzumab Fv-LIGHT fusion compared to an anti-PDl Fv-LIGHT fusion to bind to HER2; and FIG. 7C is a graph showing that the Trastuzumab Fv-LIGHT fusion maintains its ability to bind to LIGHT receptors.FIG. 8A is a diagram of a first exemplary multifunctional trimeric ligand according to the disclosure; FIG. 8B depicts SDS-PAGE and chromatograph results of the trimeric ligand; and FIGS. 8C-D are ELISA charts showing the ability of the trimeric ligand to bind to DLL3 and CD3.Docket No. BREACH-001 / WOOlFIG. 9A is a diagram of a second exemplary multifunctional trimeric ligand according to the disclosure; FIG. 9B depicts SDS-PAGE and chromatograph results of the trimeric ligand; and FIGS. 9C-F are ELISA charts showing the ability of the trimeric ligand to bind to mouse PD-L1, HSA, HVEM, and LTBR.FIG. 10A is a diagram of a third exemplary multifunctional trimeric ligand according to the disclosure; FIG. 10B depicts SDS-PAGE and chromatograph results of the trimeric ligand; FIG. 10C is an ELISA chart showing the ability of the trimeric ligand to bind to mouse PD-L1; FIG. 10D is an ELISA chart showing the ability of the trimeric ligand to bind to HSA.FIGS. 11A-F are diagrams of exemplary multifunctional trimeric ligands according to the disclosure. FIG. 11G are diagrams of multifunctional trimeric ligands used in a T cell-redirected cytotoxicity assay, and the results of the assay, where the cytotoxicity of target cells is measured. In this experiment, human PBMCs were incubated with SHP-77 cells (target cells), in the presence of DLL3 / CD3 / LIGHT fusion molecules.FIG. 12 shows the results of an ELISA assay where T cell-secreted IFN-gamma levels are measured. In this experiment, mouse PBMC were incubated for 24 hours with MC-38 in the presence or absence of an isotype control antibody, the 10F.9G2 anti-mouse PD-L1 monoclonal antibody, or the LIGHT(BB127) / PD-L1 multispecific protein that includes the Fv of 10F.9G2 and an anti-HSA VHH acting as a HLE.FIG. 13 A is a photograph of an SDS-PAGE gel and chromatograph showing wildtype CD40L; and FIGS. 13B-D are photographs of SDS-PAGE gels and chromatographs showing CD40L-based polarized trimers according to the disclosure; and FIG. 13E shows photographs photographs of SDS- PAGE gels and chromatographs showing wildtype 4-1BBL and a 4-lBBL-based polarized trimer according to the disclosure.FIG. 14 is a chart of an ELISA experiment examining the binding of various TNF trimers to the extracellular domain of human 4- IBBFIG. 15A is a cartoon describing the structure of an embodiment of a polarized LIGHT trimer fused to two anti -mouse PD-L1 VHH and to one anti -human serum albumin VHH according to the disclosure; and FIG. 15B is a diagram of the tumor volume of MC38 subcutaneous tumors in C57BL / 6-hALB / hFcRn mice upon vehicle or treatment with the polarized LIGHT trimer of FIG. 15A.DETAILED DESCRIPTIONI. OverviewDocket No. BREACH-001 / WOOlCancer is a complex and multifaceted disease. As one example, fibrotic cancers are a form of cancer that creates excessive fibrous connective tissue. In fibrotic cancers, tumor cells stimulate the surrounding tissue to produce collagen and other fibrous proteins, leading to the formation of fibrotic areas within or around the tumor mass. This fibrosis promotes tumor growth and invasion by providing structural support to tumor cells and also creates a barrier that inhibits the delivery and effectiveness of chemotherapy and other treatments. Management of these cancers often requires strategies to both mitigate fibrosis and enhance treatment delivery. Yet, most existing treatments target discrete mechanisms, limiting therapeutic efficacy and response.Accordingly, the inventor has recognized and appreciated that multi-faceted biologic agents which can address multiple interconnected pro-tumoral pathways can deliver powerful anti-cancer effects. Disclosed herein is a flexible and multi-specific platform that can include multiple functional moieties on a single molecule. The platform can be leveraged to target multiple malignant pathways simultaneously, creating multi-functional agents with the potential to significantly improve the treatment of multiple cancer types.The platform can comprise multifunctional molecules (also interchangeably referred to herein as “multispecific molecules”) that comprise a polarized trimeric ligand, e g., a trimeric ligand comprising individual monomers that will associate in a particular configuration. The trimeric ligand serves as a central connecting unit for the multifunctional molecule. In some embodiments, the multifunctional molecules have at least one, two, three, four, or more activities, e.g., binding activities and / or other functional activities. Such activities are enabled by different binding and / or functional moieties fused to individual monomers of the trimeric ligand, resulting in a highly versatile and useful platform for new drug development.In one embodiment, the polarized trimeric ligand comprises Tumor Necrosis Factor Superfamily (TNFSF) proteins. TNFSF proteins are essential for innate and adaptive immunity and are implicated in diverse pathological conditions such as autoimmune diseases, developmental abnormalities, and cancer. TNFSFs activate signaling pathways that can be beneficial to fight cancer such as T cell so-stimulation, and cancer cell apoptosis. Systemic cytokine (such as TNFSF) therapy is generally limited by rapid degradation and elimination of the cytokine, the inability to achieve optimal concentrations in the tumor, and dose-dependent toxicity, including life-threatening side effects such as vascular leak syndrome and orthostatic hypotension.TNFSF proteins, such as TNF-alpha, Fas ligand (FasL), and TRAIL often interact with one another to form trimers. This trimeric structure allows the TNFSF proteins to bind to receptors andDocket No. BREACH-001 / WOOl initiate signaling pathways involved in processes such as apoptosis, inflammation, and immune regulation. The inventor has recognized that this homotrimeric structure can be modified through protein engineering to form polarized trimers (e.g., trimers with individual monomers that associate with one another in a specific configuration) that can be fused with up to six different fusion partners at the first degree of interaction, for the creation and development of a multispecific and / or multifunctional protein with significant therapeutic utility. These multispecific / multifunctional proteins can contain a number of functional groups that confer various therapeutic properties to the protein including but not limited to: tumor targeting, half-life extension, immune cell activation, checkpoint inhibition, stromal and vascular normalization. Hence, building upon the function of the TNFSF ligand itself combined with the fusion of bespoke functional groups to the polarized TNFSF ligand trimer enable the design and production of unique and novel multifunctional proteins, with unparalleled design flexibility.Although many other multifunctional platforms are based on the engineering of IgG formats, the approach of the present disclosure is based on the engineering of TNFSF ligand templates. The inventor has recognized and appreciated that their diversity enables the design of proteins that target specific pathways, depending on the TNF ligand used as a template for a given protein.In some embodiments, the TNFSF trimeric ligand is LIGHT. LIGHT functions in both a soluble and cell surface-bound manner to interact with two primary functional receptors: herpes virus entry mediator (HVEM) and lymphotoxin- receptor (LT R). The LIGHT-HVEM interaction is responsible for enhanced T cell activation, proliferation, survival, and Thl cytokine production whereas LIGHT -LTpR signaling increases the susceptibility of cancer cells to immune responses, reconstituting chaotic tumor vasculature and supporting effector cell infiltration into tumors through providing crucial inflammatory signals such as the promotion of the formation of tertiary lymphoid structures. Accordingly, LIGHT trimers have advantageous properties that, when combined with other functional moieties as described herein, results in a multifunctional molecule that is useful for treating diseases such as cancer.In other embodiments, the polarized trimeric ligand can comprise other TNFSF ligand members, such as those which function as co-stimulators of immune cells. Several TNFSF ligands function as immune-costimulatory cytokines. Among these, 4-1BBL (TNFSF9), OX40L (TNFSF4), CD40L (TNFSF5), CD27L (TNFSF7), GITRL (TNFSF18), TL1A (TNFSF15), LTB (TNFSF3 / TNFC) and LTA (TNFSF 1) have similar properties to LIGHT and may also be used with multifunctional molecules according to the disclosure.Docket No. BREACH-001 / WOOlIn other embodiments, the polarized trimeric ligands can comprise other TNF members such as those which function as apoptosis-stimulators. Among these are FasL (TNFSF6), TRAIL (TNFSF10), and TNFA (TNFSF2). They may be used with multifunctional molecules according to the disclosure.The multifunctional molecules of the present disclosure can comprise polarized trimeric ligands. As used herein, a “trimeric ligand” refers to a molecule comprising three members, also referred to herein as “monomer molecules.” Polarized trimeric ligands refer to trimeric ligands comprising monomer molecules which associate in a specific manner. This polarization can be created by introducing specific amino acid substitutions into individual monomers.FIGS. 1 A-B illustrate the general concept of the polarized TNFSF ligand trimer. As shown in FIG. 1A, the introduction of specific mutations will promote an obligatory polarized interaction between the monomers to yield a polarized trimer. This polarized trimer becomes the basis for a fusion protein that can accommodate up to six fusion partners at the first degree of interaction. As shown in FIG. IB, the general strategy to obtain polarized trimers is to create mutations in one, two or three monomers A, B, and C. This strategy forces polarization, i.e., interaction with a specific subunit that has reciprocal / complementary mutations. For example: A interacts with B and C but not with A, or with BB, or with CC.In the case of 2 sets of mutations for each monomer (Al, A2, Bl, B3, C2, C3), the interaction will be specific, as follows:Interactions of A with B and C: Al will interact with Bl; A2 will interact with C2.Interactions of B with A and C: Bl will interact with Al; B3 will interact with C3.Interactions of C with A and B: C2 will interact with A2; C3 will interact with B3.Thus, a polarized trimeric ligand according to the disclosure comprising monomers A, B, and C will primarily associate as ABC, and not other forms such as AAA, AAB, ACA, etc. As explained in more detail below, these amino acid substitutions can result in the creation of new covalent and / or non-covalent associations between the monomers, such as disulfide bonds, salt bridges, and other mechanisms to increase the stability of the resulting polarized trimeric ligand. For example, substituting cysteine residues at certain positions in one, two, or three different LIGHT monomer molecules will result in a highly stable and polarized trimer ligand as a result of new disulfide bonds in the trimer, without the need to fuse each of the individual monomers together (e.g., N- or C-terminus of a first monomer fused to the N- or C-terminus of a second monomer).Docket No. BREACH-001 / WOOlIn some embodiments, the polarized trimeric ligand is coupled, e.g., covalently linked or fused, to one or more functional moieties. The functional moieties can be coupled to individual monomers of the trimeric ligand at one or both of the amino or carboxyl termini of the monomer. As will be explained in further detail below, such functional moieties can include tumor targeting moieties which can cause the multifunctional molecule to associate with or bind to a tumor cell. Functional moieties can also include various combinations of cytokine molecules, immune cell engagers, natural killer cell engagers, and stromal modifying moieties, which together in combination with the tumor targeting moieties help to activate the immune system against cancer or other disease.II. DefinitionsAll terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4thed., John Wiley & Sons, Inc.). All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with the disclosure.The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combinationDocket No. BREACH-001 / WOOl thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.The term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nestedDocket No. BREACH-001 / WOOl sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.The term “subject” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.The term “optional” or “optionally” denotes that a subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.“Non-naturally occurring” (used interchangeably with “isolated”, “substantially pure” or “purified”) when applied to a polynucleotide or a polypeptide means a polynucleotide or a polypeptide that is synthesized chemically or synthetically, or is purified away from associated and contaminating components. The term generally means a polynucleotide or a polypeptide that has been separated from other proteins and nucleic acids with which it naturally occurs and / or substances which are used to purify it. In some instances, polynucleotide or a polypeptide herein is about 95%, about 96%, about 97%, about 98%, about 99% or above pure.The term “antibody” or “antibody molecule” is used in its broadest sense and covers monoclonal antibodies, polyclonal antibodies, single domain antibodies, VHH, nanobodies, heavy chain-only antibodies, light chain-only antibodies, dimers, multimers, multispecific antibodies (eg. bispecific antibodies), veneered antibodies, antibody fragments and small immune proteins (SIPs) (see hit. J. Cancer (2002) 102, 75-85). An antibody is a protein generated by the immune system or obtained through other methods such as library screening or other methods of discovery that are capable of recognizing and binding to a specific antigen. A target antigen generally has numerous binding sites, also called epitopes, recognized by complementarity determining regions (CDRs) on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, ie. a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof.The term “antibody fragment” refers to a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single domain antibodies,Docket No. BREACH-001 / WOOl including dAbs, camelid and humanized VHH antibodies and the IgNAR antibodies of cartilaginous fish. Antibodies and their fragments may be replaced by binding molecules based on alternative nonimmunoglobulin scaffolds, peptide aptamers, nucleic acid aptamers, structured polypeptides comprising polypeptide loops subtended on a non-peptide backbone, natural receptors or domains thereof.An antibody “which binds” an antigen of interest is one that is capable of binding that antigen with sufficient affinity such that the antibody is useful in targeting a cell expressing the antigen. An antibody binding to a target represents the same principle.The term “molecule” as used in, e.g., an antibody molecule, a cytokine molecule, a receptor molecule, a monomer molecule, includes full-length, naturally-occurring molecules, as well as variants, e.g., functional variants (e.g., truncations, fragments, mutated (e.g., substantially similar sequences) or derivatized form thereof), so long as at least one function and / or activity of the unmodified (e.g., naturally-occurring) molecule remains. In an embodiment, the molecule comprises at least 70 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent or higher sequence identity to a naturally-occurring molecule.The term “functional variant” refers to polypeptides that have a substantially identical amino acid sequence to the naturally-occurring sequence, or are encoded by a substantially identical nucleotide sequence, and are capable of having one or more activities of the naturally-occurring sequence.“Derived from” as used herein in reference the relationship of a first sequence, to a second sequence (e.g., in the context of nucleic acid sequence or protein sequences) imposes no process limitations and refers only to structural similarity. In embodiments a derived sequence will differ from the reference sequence by levels of homology or sequence identity described elsewhere herein.“Trimeric ligand” as used herein refers to a molecule comprising three members, also referred to herein as “monomer molecules.” In embodiments, the three members are three covalently or non- covalently associated polypeptides. In some embodiments, the trimeric ligand comprises three non- covalently associated monomer molecules. In one embodiment, the trimeric ligand comprises two covalently associated monomer molecules, e.g., joined by a disulfide bond introduced by specific amino acid changes and one non-covalently associated monomer molecule. In yet other embodiments, the trimeric ligand comprises three covalently associated monomer molecules, e.g., joined by two or more disulfide bonds introduced by specific amino acid changes. In embodiments wherein two monomer molecules are covalently linked monomers, the linked monomers can be reading from leftDocket No. BREACH-001 / WOOl to right have the following configuration: Amino terminal to carboxy terminal, or carboxy terminal to amino terminal. In embodiments, the trimeric ligand interacts, e.g., binds to a target molecule, e.g., a receptor.In some embodiments, the multifunctional molecule includes a tumor-targeting moiety. A “tumor-targeting moiety,” as used herein, refers to a binding agent that recognizes or associates with, e.g., binds to, a target in a cancer cell. The tumor-targeting moiety can be an antibody molecule, a receptor molecule (e.g., a full length receptor, receptor fragment, or fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule (e g., a full length ligand, ligand fragment, or fusion thereof (e.g., a ligand-Fc fusion)) that binds to the cancer antigen (e.g., the tumor and / or the stromal antigen). In embodiments, the tumor-targeting moiety specifically binds to the target tumor, e.g., binds preferentially to the target tumor. For example, when the tumor-targeting moiety is an antibody molecule, it binds to the cancer antigen (e.g., the tumor antigen and / or the stromal antigen) with a dissociation constant of less than about 10 nM, and more typically, 10-100 pM.In some embodiments, the multifunctional molecule includes an immune cell engager. An “immune cell engager” refers to one or more binding specificities that bind and / or activate an immune cell, e g., a cell involved in an immune response. In embodiments, the immune cell is chosen from an NK cell, a T cell, a B cell, a dendritic cell, and / or the macrophage cell. The immune cell engager can be an antibody molecule, a receptor molecule (e.g., a full length receptor, receptor fragment, or fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule (e.g., a full length ligand, ligand fragment, or fusion thereof (e.g., a ligand-Fc fusion)) that binds to the immune cell antigen (e.g., the NK cell antigen, the T cell antigen, the B cell antigen, the dendritic cell antigen, and / or the macrophage cell antigen). In embodiments, the immune cell engager specifically binds to the target immune cell, e.g., binds preferentially to the target immune cell. For example, when the immune cell engager is an antibody molecule, it binds to the immune cell antigen (e.g., the NK cell antigen, the T cell antigen, the B cell antigen, the dendritic cell antigen, and / or the macrophage cell antigen) with a dissociation constant of less than about 10 nM, and more typically, 10-100 pM.In some embodiments, the multifunctional molecule includes a cytokine molecule. As used herein, a “cytokine molecule” refers to full length, a fragment or a variant of a cytokine; a cytokine further comprising a receptor domain, e.g., a cytokine receptor dimerizing domain; or an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) to a cytokine receptor, that elicits at least one activity of a naturally-occurring cytokine. In some embodiments the cytokine molecule is chosen from interleukin-2 (IL -2), interleukin-4 (IL-4), interleukin- 10 (IL-10), interleukin-Docket No. BREACH-001 / WOOl12 (IL-12), interleukin- 15 (IL-15), interleukin- 18 (IL-18), interleukin-21 (IL-21), or interferon gamma, or a fragment or variant thereof, or a combination of any of the aforesaid cytokines. The cytokine molecule can be a monomer molecule or a dimer. In embodiments, the cytokine molecule can further include a cytokine receptor dimerizing domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) to a cytokine receptor chosen from an IL-2R, IL-15Ra or IL-21R.“Modification” herein is meant an amino acid substitution, insertion, and / or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a protein. For example, a modification may be an altered carbohydrate or PEG structure attached to a protein. By “amino acid modification” herein is meant an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. For clarity, unless otherwise noted, the amino acid modification is always to an amino acid coded for by DNA, e g., the 20 amino acids that have codons in DNA and RNA.“Amino acid substitution” or “substitution” herein means the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism. For example, the substitution E272Y or 272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced with tyrosine. For clarity, a protein which has been engineered to change the nucleic acid coding sequence but not to change the starting amino acid (for example exchanging CGG (encoding arginine) to CGA (still encoding arginine) to increase host organism expression levels) is not an “amino acid substitution”; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution.“Amino acid insertion” or “insertion” as used herein means the addition of an amino acid residue or sequence at a particular position in a parent polypeptide sequence. For example, -233E designates an insertion of glutamic acid after position 233 and before position 234. Additionally, - 233ADE or A233ADE designates an insertion of AlaAspGlu after position 233 and before position 234.“Amino acid deletion” or “deletion” as used herein means the removal of an amino acid residue or sequence at a particular position in a parent polypeptide sequence. For example, E233-, E233#, E233(), E233_, or E233del designates a deletion of glutamic acid at position 233. Additionally, EDAZ33- or EDA2331designates a deletion of the sequence GluAspAla that begins at position 233.Docket No. BREACH-001 / WOOl“Variant protein”, “protein variant”, “mutein”, or “variant” as used herein means a protein that differs from that of a parent protein by virtue of at least one modification. Protein variant may refer to the protein itself, a composition comprising the protein, the amino acid sequence that encodes it, or the DNA sequence that encodes it Preferably, the protein variant has at least one amino acid modification compared to the parent protein, e g. from about one to about seventy amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent, The modification can be an addition, deletion, or substitution. As described below, in some embodiments the parent protein, for example an Fc parent polypeptide, is a human wild type sequence, such as the Fc region from IgGl, IgG2, IgG3 or IgG4. The protein variant sequence herein will preferably possess at least about 80% identity with a parent protein sequence, and most preferably at least about 90% identity, more preferably at least about 95-98-99% identity. “Variant,” as used herein can also refer to particular amino acid modifications (e g, substitutions, deletions, insertions) in a variant protein (e.g., a variant Fc domain), for example, heterodimerization variants, ablation variants, etc.As used herein, “Protein” means at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. When a biologically functional molecule comprises two or more proteins, each protein may be referred to as a “monomer” or as a “subunit”; and the biologically functional molecule may be referred to as a “complex”. Accordingly, “protein” in this context is used interchangeably with “polypeptide” and includes peptides as well.“Residue” as used herein means a position in a protein and its associated amino acid identity. For example, Asparagine 297 (also referred to as Asn297 or N297) is a residue at position 297 in the human antibody IgGl.“Non-naturally occurring modification” as used herein with respect to an IgG domain means an amino acid modification that is not isotypic. For example, because none of the IgGs comprise a serine at position 434, the substitution 434S in IgGl, IgG2, IgG3, or IgG4 (or hybrids thereof is considered a non-naturally occurring modification.“Amino acid” and “amino acid identity” as used herein means one of the 20 naturally occurring amino acids that are coded for by DNA and RNA.“Effector function” as used herein means a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include but are not limited to ADCC, ADCP, and CDC.Docket No. BREACH-001 / WOOl“Parent polypeptide” as used herein means a starting polypeptide that is subsequently modified to generate a variant. The parent polypeptide may be a naturally occurring polypeptide (i.e., a wildtype polypeptide), or a variant or engineered version of a naturally occurring polypeptide. Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it.“Fusion protein” as used herein means the covalent joining of at least two proteins or protein domains. Fusion proteins may comprise artificial sequences, e.g. a domain linker, an Fc domain (e.g., a variant Fc domain), a variant TNFSF ligand (e.g., a variant LIGHT), a functional protein, etc. as described herein. By “Fc fusion protein” or “immunoadhesin” herein is meant a protein comprising an Fc region, generally linked (optionally through a domain linker, as described herein) to one or more different protein domains. Accordingly, an “LIGHT fusion protein” includes a functional domain linked (optionally through a domain linker) to a mutated or wildtype LIGHT monomer, as described herein.“Position” as used herein means a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the EU index for numbering of antibody domains (e.g., a CHI, CH2, CH3 or hinge domain).“Strandedness” in the context of the monomers of the heterodimeric proteins of the disclosure herein means that, similar to the two strands of DNA that “match”, heterodimerization variants are incorporated into each monomer so as to preserve, create, and / or enhance the ability to “match” to form heterodimers. For example, if some pl variants are engineered into monomer A (e.g. making the pl higher), then steric variants that are “charge pairs” that can be utilized as well do not interfere with the pl variants, e.g., the charge variants that make a pl higher are put on the same “strand” or “monomer” to preserve both functionalities. Similarly, for “skew” variants that come in pairs of a set as more fully outlined below, the skilled artisan will consider pl in deciding into which strand or monomer that incorporates one set of the pair will go, such that pl separation is maximized using the pl of the skews as well.“Wild type,” “wildtype”, “wild-type”, or “WT” herein means an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A WT protein has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.The various proteins, antibodies, binding proteins, and fusion proteins provided herein are generally isolated or recombinant. “Isolated,” when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and / or recovered from aDocket No. BREACH-001 / WOOl cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An “isolated protein” refers to a protein which is substantially free of other proteins from a cell culture such as host cell proteins. “Recombinant” means the proteins are generated using recombinant nucleic acid techniques in exogeneous host cells.“Percent (%) amino acid sequence identity” with respect to a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific (parental) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. One particular program is the ALIGN-2 program outlined at paragraphs

[0279] to

[0280] of US Patent Publication No. 20160244525, hereby incorporated by reference.The degree of identity between an amino acid sequence provided herein (“disclosure sequence”) and the parental amino acid sequence is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the “disclosure sequence,” or the length of the parental sequence, whichever is the shortest. The result is expressed in percent identity. The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller Compiit. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol., 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GGG software package (available commercially), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3,4, 5, or 6.In some embodiments, two or more amino acid sequences are at least 50%, 60%, 70%, 80%, or 90% identical. In some embodiments, two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical. In general, the percentage identity for comparison between amino acid sequences is at least 75%, at least 80%, at least 90%, with at least about 95, 96, 97, 98 or 99%Docket No. BREACH-001 / WOOl percent identity being preferred. The percentage identity may be along the whole amino acid sequence, for example the entire protein sequence.“Fused” or “covalently linked” means that the components (e g., an TNF ligand monomer and a VHH) are linked by peptide bonds, either directly or indirectly via domain linkers, as outlined herein. The strength, or affinity, of specific binding can be expressed in terms of the dissociation constant (KD) of the interaction, wherein a smaller KD represents greater affinity, and a larger KD represents lower affinity. Binding properties can be determined by methods well known in the art such as biolayer interferometry (BLI) and surface plasmon resonance-based methods (SPR). One such method entails measuring the rates of antigen-binding site / antigen or receptor / ligand complex association and dissociation, wherein rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the association rate (ka) and the dissociation rate (kd) can be determined, and the ratio of kd / ka is equal to the dissociation constant KD (See Nature 361.186-187 (1993) and Davies et al. (1990) Annual Rev Biochem 59:439-473).Specific binding for a particular molecule or an epitope can be exhibited, for example, by a molecule (e.g., LIGHT) having a KD for its binding partner (HVEM or LTbetaR) of at least about 10"4M, at least about 10‘5M, at least about 10’6M, at least about 10'7M, at least about 10‘8M, at least about 10'9M, alternatively at least about IO'10M, at least about 10-11M, at least about 10'12M, or greater. Typically, an antigen binding molecule that specifically binds an antigen will have a KD that is 20, 50, 100, 500, 1000, 5,000, 10,000 or more times greater for a control molecule relative to the antigen or epitope.III. Trimeric Ligand Platform for Multispecific and / or Multifunctional MoleculesAs previously noted, cancer is a complex and multifaceted disease. For example, fibrotic cancer relies on multiple intricate molecular mechanisms and interactions between diverse cell types. Most existing treatments target discrete mechanisms, resulting in poor therapeutic response. In contrast, a multifaceted biologic that addresses several interconnected pro-tumoral pathways can deliver impactful anti-cancer effects.Embodiments of the disclosure can attack cancer by targeting multiple malignant pathways simultaneously. Such embodiments can assemble multiple distinct functional groups into a single multifunctional and / or multi-specific molecule. For example, a trimeric ligand can have a functional group and central connector (the trimeric ligand itself); up to three proximal functional groups (e.g.,Docket No. BREACH-001 / WOOl fused to the N-termini of individual monomers within the trimeric ligand); and up to three distal functional groups (e.g., fused to the C-termini of the individual monomers).Such molecules can be engineered to target several mechanisms of resistance. For example, T cell anti-tumor activity depends on their ability to migrate to and penetrate a tumor and then kill cancer cells. As described in more detail herein, embodiments of the disclosure can combine several complementary mechanisms of action to combat cancer (e.g., fibrotic cancer) including tumor trafficking, tumor targeting, T cell engagement and co-stimulation, vasculature remodeling, and checkpoint inhibition. For example, a multi-functional T cell infiltrator and engager can comprise a trimeric ligand that includes a tumor targeting moiety, a checkpoint inhibitor, a T cell infiltrator, a T cell engager, and a central unit comprising an immune cell co-stimulator and vasculature remodeler, as described further herein.In some aspects described herein are multifunctional molecules comprising a trimeric ligand. The trimeric ligand can comprise a first monomer molecule, a second monomer molecule, and a third monomer molecule. In some aspects, the first, second, and / or third monomer molecules comprise TNFSF ligand mutants. Such TNFSF ligand mutants contain certain mutations compared to their wildtype counterparts that enable the formation of polarized trimers, i.e., each of the first, second, and third monomer molecules associate with one another predominantly in a specific configuration to create a trimeric ligand. As described in further detail herein, such mutations were selected to increase the stability, expression yield, and overall utility of the resulting polarized trimeric ligand. Additional benefits may be conferred by fusing such polarized trimeric ligands with selected functional fusion partners, including but not limited to TAA binders, tumor targeting moieties, immune targeting moieties, checkpoint modulators, stromal and vascular normalizers, immune stimulators, half-life extensions, and enzymes. Accordingly, embodiments of the disclosure can be used to create a variety of therapeutic molecules with both multispecific and multifunctional characteristics.(a) Trimeric LigandsA trimeric ligand can comprise three members, also referred to herein as “monomer molecules.” In embodiments, the three members are three covalently or non-covalently associated polypeptides. In some embodiments, the trimeric ligand comprises three non-covalently associated monomer molecules. In one embodiment, the trimeric ligand comprises two covalently associated monomer molecules, e.g., joined by a disulfide bond introduced by specific amino acid changes introduced into two of the monomer molecules and one non-covalently associated monomer molecule. In yet other embodiments, the trimeric ligand comprises three covalently associated monomerDocket No. BREACH-001 / WOOl molecules, e.g., joined by two or more disulfide bonds introduced by specific amino acid changes introduced into the three monomer molecules. In embodiments wherein two monomer molecules are covalently linked monomers, the linked monomers reading from left to right have the following configuration: Amino terminal to carboxy terminal, or carboxy terminal to amino terminal. In embodiments, the trimeric ligand interacts, e.g., binds to a target molecule, e.g., a receptor for the trimeric ligand (such as LIGHT and its receptors HVEM and LT R). Therefore, in addition to constituting the basis for a multifunctional / multispecific platform, the polarized TNFSF trimeric ligands are endowed with their biological activity (i.e., T cell co-stimulation, NEC cell activation, dendritic cell maturation, stimulation of angiogenesis, and stromal normalization in the case of LIGHT), representing an ideal platform for developing new targeted therapeutics for cancer and other diseases.The trimeric ligand can comprise both covalent and non-covalent associations to increase its stability and to promote a specific configuration between the individual monomers. In some embodiments, the covalent associations comprise one or more disulfide bonds. The one or more disulfide bonds can be created by introducing new cysteine residues at certain positions in each monomer, thus linking the monomers to one another in a specific configuration. In some embodiments, the covalent associations comprise one or more isopeptide bonds. The one or more isopeptide bonds can be created by introducing new lysine, glutamine, or asparagine residues at certain positions in each monomer to facilitate the isopeptide bond formation. In some embodiments, the non- covalent associations comprise one or more salt bridges. The one or more salt bridges can be created similarly introduced by residue changes in each monomer, e.g., positively charged residues (lysine, arginine, histidine) in one monomer and negatively charged residues (aspartate, glutamate) in another. In some embodiments, the non-covalent associations can comprise one or more hydrogen bonds created by substituting residues with hydroxyl or amide side chains (like serine, threonine, asparagine, or glutamine) that can promote hydrogen bonding between two monomer molecules. In some embodiments, the non-covalent associations can comprise hydrophobic interactions created by substituting polar or charged residues with nonpolar ones (leucine, isoleucine, valine, phenylalanine) to create hydrophobic patches that promote association between polypeptides. In some embodiments, the non-covalent associations can comprise Pi-Pi interactions by substituting non-aromatic residues with aromatic residues such as phenylalanine, tyrosine, histidine, and tryptophane. In some embodiments, the non-covalent associations can comprise van der Waals interactions, e.g., byDocket No. BREACH-001 / WOOl introducing smaller residues like glycine or larger nonpolar residues like leucine. Various non- covalent associations introduced by specific residue changes (e.g., aromatic, cation, and electrostatic interactions) are considered within the scope of the disclosure.In some embodiments, the stability of the trimeric ligand can be increased by substituting phenylalanine for tyrosine at specific residues, e.g., to create new hydrogen bonds between the monomers. Such amino acid changes, in combination with the introduced covalent and / or non- covalent associations described herein, may further increase stability of the trimeric ligand.In some embodiments, the trimeric ligand is a member of the TNF superfamily, also referred to herein as a “TNFSF family member.” The tumor necrosis factor (TNF) superfamily refers to a superfamily of cytokines identified as part of the TNF family on the basis of, e.g., sequence, function and / or structural similarities, e.g., reviewed in Sun M, Fink PJ (2007) J Immunol. 179 (7): 4307-12; Peitsch MC, Jongeneel CV (1993) Int. Immunol. 5 (2): 233-8; Farrah T, Smith CA (July 1992) Nature 358 (6381): 26; Bazan JF (1993). Curr. Biol. 3 (9): 603-6. TNFSF family members typically form homotrimeric or heterotrimeric complexes.In embodiments, the trimeric ligand is chosen from a TNFSF family member or a TNF-like family member, or a combination thereof, e.g., as described herein in Tables 1 and 2, below. In some embodiments, the trimeric ligand is a homotrimer, e.g., it comprises three monomer molecules from the same trimeric ligand e.g., the same TNFSF family member or the same TNF-like family member, in which one or more of the monomers can comprise mutations that create covalent and / or non- covalent associations, as further described herein. In other embodiments, the trimeric ligand is a heterotrimer, e.g., it comprises a combination of monomer molecules from two or three trimeric ligands, e.g., two or three TNFSF or TNF-like family members, in which one or more of the monomers can comprise mutations that create covalent and / or non-covalent associations, as further described herein.In one embodiment, the trimeric ligand is a heterotrimer comprising TNFSF12 and TNFSF13 monomer molecules (e.g., TWE-PRIL17). In some embodiments, the trimeric ligand can comprise one TNFSF12 monomer molecule and two TNFSF13 monomer molecules. In some embodiments, the trimeric ligand can comprise two TNFSF12 monomer molecules and one TNFSF13 monomer molecule. In such embodiments, the monomers can comprise mutations that create new covalent and / or non-covalent associations between monomers, as described herein.Docket No. BREACH-001 / WOOlTable 1: Wild-type sequences for TNFSF family membersDocket No. BREACH-001 / WOOlTable 2: Wild-type sequences for TNF-like superfamily membersIn embodiments, the trimeric ligand comprises the amino acid sequence of a monomer molecule chosen from a TNFSF family member or a TNF-like family member, e.g., as described herein in Tables 1 and 2, in which some of the monomers (e.g., at least one, at least two, or at least three) include certain mutations that result in new covalent or non-covalent associations within the trimeric ligand as further described herein. In some embodiments, the trimeric ligand comprises an amino acid sequence substantially identical to a TNFSF family member or a TNF-like family member, e.g., at least 85%, 90%, 95%, 99% or more identical to an amino acid described in Table 1, in which some of the monomers (e.g., at least one, at least two, or at least three) include certain mutations that result in new covalent or non-covalent associations within the trimeric ligand as further described herein. For example, the trimeric ligand can comprise any of the TNFSF amino acid sequences for TNSF1, TNSF2, TNSF3, TNSF4, TNSF5, TNSF6, TNSF7, TNSF8, TNSF9, TNSF10, TNSF11, TNSF12, TNSF12, TNSF13, TNSF13B, TNSF14, TNSF15, TNF18 or EDA, corresponding to SEQ ID NOs: 1-17, in Table 1, or an amino acid sequence substantially identical thereto, e.g., at least 85%, 90%, 95%, 99% or more identical to the amino acid sequence of any of SEQ ID NOs: 18-26, as a homotrimer, or a heterotrimer comprising any combination of two or three of the aforesaid monomer molecules, in which some of the monomers (e.g., at least one, at least two, or at least three) include certain mutations that result in new covalent or non-covalent associations within the trimeric ligand as further described herein. In some embodiments, the trimeric ligand can comprise any of the TNF- like amino acid sequences for Complement C1Q (e.g., subcomponents A, B and C), C1QL1, C1QL2, C1QL3, Caprin-2 Clq domain, cerebellin-1 Clq domain or adiponectin, corresponding to SEQ ID NOs: 18-26, in Table 2, or an amino acid sequence substantially identical thereto, e.g., at least 85%, 90%, 95%, 99% or more identical to the amino acid sequence of any of SEQ ID NOs: 18-26, as a homotrimer (e.g., comprises of homomonomer molecules), or a heterotrimer (e.g., comprises of heteromonomer molecules) comprising any combination of two or three of the aforesaid monomers, in which some of the monomers (e.g., at least one, at least two, or at least three) include certain mutations that result in new covalent or non-covalent associations within the trimeric ligand as further described herein. In other embodiments, the trimeric ligand comprises a combination of TNFSF monomer molecules and TNF-like monomer molecules, wherein the trimer ligand is a heterotrimer comprising any combination of two or three monomer molecules comprising the amino acid sequenceDocket No. BREACH-001 / WOOl of any of SEQ ID NOs: 1-26, in which some of the monomers (e.g., at least one, at least two, or at least three) include certain mutations that result in new covalent or non-covalent associations within the trimeric ligand as further described herein.In some embodiments, the tumor necrosis factor superfamily member is chosen from one or more of the sequences in Tables 1 or 2.Embodiments of the disclosure comprise trimeric ligands with new functional properties created by utilizing variant TNFSF family proteins. The variant TNFSF proteins result in changes to the trimeric ligand that can increase stability (useful for protein expression and function) and also promote a particular configuration of the trimeric ligand. Because the trimeric ligand forms a particular configuration, it may be utilized as a base unit for a fusion protein molecule, e.g., by fusing various functional moieties to individual variant TNFSF proteins to create a multispecific and / or multifunctional molecule, as further described herein.In some embodiments, a trimeric ligand comprises a first monomer, a second monomer, and a third monomer, wherein two or more of the monomers comprise amino acid substitutions compared to wild-type that result in a new disulfide bond between the monomers. In some embodiments, the trimeric ligand comprises 1, 2, 3, 4, 5, 6, or more new disulfide bonds resulting from amino acid substitutions within individual monomers. In some embodiments, there are up to 2 disulfide bonds connecting each monomer to one another within a trimeric ligand. For example, a first monomer can contain two mutations that form disulfide bonds with second and third monomers that have corresponding mutations. The second and third monomers may further form an additional disulfide bond due to additional mutations within those monomers. Similarly, some or all of the disulfide bonds described above could be replaced with salt bridges or other non-covalent associations introduced through mutations within individual monomers.In some embodiments, the positions of residues that are mutated within individual monomers are selected based on their proximity to positions of residues in other monomers within the trimeric ligand. Preferably, the positions selected are those in which the atoms within each residue that engage in inter-chain or inter-monomer interactions are within 1, 2, 3, 4, 5, or 6 angstroms from one another. Such positions are ideal for substituting residues with different amino acids that can create covalent and / or non-covalent associations within the trimeric ligand and promote a particular configuration.Embodiments of the disclosure comprise TNFSF ligands. In some embodiments that are based on variant LIGHT (TNFSF 14) proteins, the modifications can include amino acid substitutions at one or more of the following positions: N93, R124, Y142, Y144, S182, Q183, S185, G188, R189, T191,Docket No. BREACH-001 / WOOlR195, S200, F202, R232, F238, and V240. In some embodiments, the variant LIGHT protein can further comprise an additional C-terminal extension sequence of R241X242X243D244, X241X242X243X244, X241X242X243. or X241X242, wherein X242 or X243 comprise a cysteine residue. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both positions can be modified to create a new covalent or non-covalent association, as further described herein: N93 / X242, N93 / X242, S182 / S200, G188 / T191, R124 / V240; Q183 / R232; R189 / R195; and S185 / R195. If using R124 / V240 mutations to form a salt bridge between the two residues R124 may be wild-type, whereas V240 may be modified to aspartic acid (V240D). In some embodiments, the modifications can comprise an amino acid substitution of a phenylalanine residue for a tyrosine residue at one or more position so as to add a hydroxyl group that forms new hydrogen bonds within and between the monomers, as described herein: F202Y, F238Y.In some embodiments that are based on variant CD27L proteins, the modifications can include amino acid substitutions at one or more of the following positions: SI 17 and Q149. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both positions can be modified to create a new covalent or non-covalent association, as further described herein: SI 17; and Q149. In such embodiments, the modifications can include amino acid substitutions at the same positions in other monomers so as to promote the formation of a new covalent or non-covalent association as described herein (e.g., S117W, Q149Y).In some embodiments that are based on variant CD40L proteins, the modifications can include amino acid substitutions at one or more of the following positions: C178, C194, A208, T211, S213, C218, G219, and S222. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both of the positions can be modified to create a new covalent or non-covalent association, as further described herein: A208 / S222; T211 / S213; and T211 / G219. In some embodiments, the modifications can comprise an amino acid substitution of a cysteine residue to a serine residue at one or more positions so as to avoid the undesired formation of disulfide bonds and to increase the stability of the trimeric ligand, as further described herein: C178S, C194S, and C218S.In some embodiments that are based on variant TL1A proteins, the modifications can include amino acid substitutions at one or more of the following positions: R96, D146, F148, C163, M197, C203, D242, and F244. In such embodiments, the following pairs of positions may be sufficientlyDocket No. BREACH-001 / WOOl close to one another between a first monomer and a second monomer in the trimeric ligand that one or both of the positions can be modified to create a new covalent or non-covalent association, as further described herein: M197 / D242; and R96 / D146. In some embodiments, the modifications can comprise an amino acid substitution of a cysteine residue to a serine residue at one or more positions so as to avoid the undesired formation of disulfide bonds and to increase the stability of the trimeric ligand, as further described herein: C163S, C203S. In some embodiments, the modifications can comprise an amino acid substitution of a phenylalanine residue for a tyrosine residue at one or more position so as toadd a hydroxyl group that forms new hydrogen bonds within and between the monomers, as described herein: F148Y, F244Y.In some embodiments that are based on variant LTB proteins, the modifications can include amino acid substitutions at one or more of the following positions: L138, G181, and S204. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both of the positions can be modified to create a new covalent or non-covalent association, as further described herein: G181 / S204; and L138 / G181.In some embodiments that are based on variant LTA and / or variant LTB proteins (e.g., a heterotrimeric LTA-LTB-LTB complex), the modifications can include amino acid substitutions at one or more of the following positions in LTA: M154 and L164; and in LTB: R160, G191, A192, Y212, and S214. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both of the positions can be modified to create a new covalent or non-covalent association, as further described herein: LTB:A192 / LTB:Y212, LTA:M154 / LTB:Y212, and LTB: Al 92, LTB: Y212.In some embodiments that are based on variant 4-1 BBL proteins, the modifications can include amino acid substitutions at one or more of the following positions: G80, D184, R193, and T241C. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both of the positions can be modified to create a new covalent or non-covalent association, as further described herein: D814 / R193; and G80 / T241.In some embodiments that are based on variant TRAIL proteins, the modifications can include amino acid substitutions at one or more of the following positions: D203, C230, and S232. In such embodiments, the following pairs of positions may be sufficiently close to one another between a first monomer and a second monomer in the trimeric ligand that one or both of the positions can beDocket No. BREACH-001 / WOOl modified to create a new covalent or non-covalent association, as further described herein: D203, and S232. In some embodiments, the modifications can comprise an amino acid substitution of a cysteine residue to another residue to promote the formation of a disulfide bond between two other residues, as further described herein: C230.In some embodiments, the mutations within individual monomers are selected based on their ability to increase the stability of the trimeric ligand, e.g., by substituting hydrophobic residues at positions which form the interface between the monomers within the trimeric ligand. For example, in a LIGHT (TNFSF14) trimeric ligand, the following position is suitable for modification within individual monomers: F202, e.g., F202Y. Without wishing to be bound by theory, the creation of new covalent and / or non-covalent associations within a trimeric ligand may cause the monomers within the ligand to bind closer and tighter than they would in a wild-type molecule. In some cases, this may cause the monomers to fail to associate into a trimer due to residues with relatively large side chains (such as F202) that point inwards towards the cavity. However, by replacing the residues at these positions with residues having hydroxyl groups, new hydrogen bonds can form within and between the monomers, thus stabilizing the ligand. For example, and with reference to FIG. 2, substituting phenylalanine for tyrosine at a specific position (e.g., F202Y) in each monomer adds a hydroxyl group that forms new hydrogen bonds within and between the monomers, which in combination with the introduction of newly introduced covalent and / or non-covalent associations as described herein, may further increase stability of the trimeric ligand. Other possible substitutions include: F238Y, as compared to wild-type LIGHT; and F148Y and F244Y, as compared to wild-type TL1 A.In some embodiments, the mutations within individual monomers are selected based on their ability to increase the stability of the trimeric ligand, e.g., by promoting the association between certain monomers through the stabilization of specific interactions (and by preventing certain residues from forming undesired associations that could prevent the creation of new covalent and / or non- covalent associations as described herein). For example, in a LIGHT (TNFSF14) trimeric ligand, a new disulfide bond can be created between monomers by creating paired amino acid substitutions at positions G188C (monomer B) and T191C (monomer C). This new disulfide bond can be stabilized by creating a network of electrostatic interactions around and including the disulfide bond. Such a network can be achieved by the replacement of SI 85 with a E and of R189 with aD (S185E, R189D). These new acidic residues on monomer B are intended to establish electrostatic interactions with R195 on monomer C which in turn may interact with, and stabilize, the disulfide bond. However, this interaction can be hindered due to the potential interaction between D189 (monomer B) and E185Docket No. BREACH-001 / WOOl(monomer A) and R195. By mutating R195D in monomer A, this potential interaction is prevented, forcing monomers B and C to interact and form a disulfide bond between S188C and S191C (e.g., as shown in BB241 in Table 3). (Similarly, G188 and T191 could be modified to create other covalent and / or non-covalent associations, such as salt bridges, as described herein.)In some embodiments, the mutations within individual monomers are selected based on their ability to prevent undesired aggregates of the monomers, e.g., configurations of the monomers A, B, C that are not polarized, or that otherwise form undesired oligomers or non-functional trimers. In some embodiments, positions having serine residues that could interact with newly introduced cysteine residues are replaced with alanine. For example, the serine residues could form strong hydrogen bonds with the cysteine residues and lead to undesired oligomers. By replacing these residues with alanine, there is no hydroxy group to form hydrogen bonds with cysteine, thus promoting the desired configuration of the trimeric ligand. In embodiments comprising LIGHT monomers, such positions include: SI 82, S200, e.g., S182A, S200A.In some embodiments, positions having residues which do not form covalent or non-covalent associations in wild-type trimers (such as unpaired cysteines) are mutated to prevent undesired associations forming from newly introduced modifications in the trimeric ligand. For example, unpaired cysteine residues can be replaced with structurally similar residues, such as serine. Such modifications will prevent these residues from interacting with (e.g.) newly introduced cysteine residues in other monomers. For example, in TL1A trimeric ligands, such modifications can include positions C203, C163, e.g., C203S, C163S. Similarly, wild-type residues which may form salt bridges with newly introduced aspartic acid residues may be substituted with, e.g., alanine residues to prevent hydrogen bonds from forming. For example, in LIGHT trimeric ligands, a modification at position V240 (e.g., V240D) in one monomer can result in salt bridges forming with wild-type R124 in the other monomers, resulting in undesired oligomers. Such undesired oligomers can be avoided by mutating residues at such positions to (e.g.) alanine residues, e.g., R124A or leucine residues, e.g., R124L. Thus, the V240D mutation in one monomer will only pair with the wild-type R124 residue in another monomer, whereas the other two R124 residues in the trimeric ligand are mutated to alanine (R124A) or leucine (R124L).In some embodiments, the trimeric ligand comprises monomers that are fused to one another, e.g., a first monomer having an N-terminus or C-terminus fused to an N-terminus or C-terminus of a second monomer. Such embodiments may help promote a particular configuration of the trimericDocket No. BREACH-001 / WOOl ligand; however, they may also reduce the number of functional moieties that may be fused to the trimeric ligand, as two possible termini for such units are in use.In some embodiments, the trimeric ligand comprises a combination of modifications that are selected based on proximity to one another, that increase the stability of the trimeric ligand, and prevent undesired aggregates or unintended associations.In some embodiments, one of the monomer molecules may be a wild-type molecule. In such embodiments, the wild-type molecules may form trimers separate from desired polarized trimer (e.g., where A and B are mutated monomer molecules and C is wild-type, polarized trimer ABC may form, but trimers CCC may also be present). One way to address this issue is to make sure that a tag that will be used for protein purification is fused to a monomer engaged in a disulfide bond. In that case, trimers formed with the wildtype monomers will not be purified (they will be eliminated during the purification step). Since the monomers can only associate to form trimers, each covalently bound dimer will interact with a wildtype monomer.In some embodiments, one or more of the monomer molecules can further comprise a C -terminal extension sequence. In some embodiments, the C-terminal extension sequence can comprise an amino acid sequence of X1X2X3X4, X1X2X3, or X1X2, wherein X2 or X3 comprise a cysteine residue (wherein the numbering 1, 2, 3, 4 here refers to positions after the C-terminus of the monomer molecule). In some embodiments, the amino acid sequence comprises a C-terminal sequence of TNFSF11 (RANKL): RDID, wherein the C-terminal sequence is modified to become either: RCXD (e.g., RCID) or RXCD (e.g., RDCD). In such embodiments, a new cysteine residue introduced via the C-terminal extension sequence can form a new disulfide bond with a cysteine residue in one or more of the monomer molecules. For example, in embodiments of trimeric ligands comprising LIGHT, a C-terminal extension sequence of X1X2X3X4, X1X2X3, or X1X2, wherein X2 or X3 comprise a cysteine residue (e.g., RCI, RDC, RCID, or RDCD) will form a disulfide bond with a monomer comprising a N93C mutation. Note that the use of positions 1, 2, 3, 4 in such embodiments refers to the positions of the C-terminal extension (i.e., the first position of the C-terminal extension is “1”). However, in embodiments for specific TNFSF ligands, the numbering used for the C-terminal extension can comprise the length of the TNFSF ligand itself. For example, in embodiments of trimeric ligands comprising LIGHT, a C-terminal extension sequence can comprise R241X242X243D244, X241X242X243X244, X241X242X243, or X241X242, wherein X242orX243 comprise a cysteine residue (wherein the numbering 241, 242, 243, 244 refers to positions in a modified LIGHT molecule comprising the C-terminal extension, the C-terminal extension beginning at position 241).Docket No. BREACH-001 / WOOlIn one embodiment, a trimeric ligand comprises a first monomer, a second monomer, and a third monomer. In some embodiments, the monomers each comprise a first, second, and third variant LIGHT protein. Each of the variant LIGHT proteins can comprise a modification at one or more amino acid positions selected from the group consisting of N93, R124, Y142, Y144, S182, Q183, S185, G188, R189, T191, R195, S200, F202, R232, F238, and V240, as compared to wild-type LIGHT. In some embodiments, the variant LIGHT protein can further comprise an additional C-terminal extension sequence of X241X242X243X244, X241X242X243, or X241X242, wherein X242 or X243 comprise a cysteine residue. In some embodiments, new cysteine residues are introduced into the monomers, resulting in new disulfide bonds forming between individual monomers in the trimeric ligand. This increases stability and promotes a particular configuration of the monomers in the trimeric ligand. In some embodiments, residues are modified to create non-covalent associations, such as salt bridges, between monomers for similar purposes. In some embodiments, other modifications are included to strengthen the interaction between monomers. In some embodiments, the trimeric ligand comprises one or more monomers described in Table 3, below. Various combinations of individual monomers including specific mutations, including additional modifications that may create disulfide bonds, increase stability, or prevent undesired oligomers are provided in Table 3, below.Docket No. BREACH-001 / WOOlDocket No. BREACH-001 / WOOlDocket No. BREACH-001 / WOOlDocket No. BREACH-001 / WOOlTable 3: Exemplary Trimeric Ligands based on Combinations of Mutated and Wild-Type LIGHT Monomers. Mutations D242C and I243C (e.g., in BB287-BB362) refer to a monomer further comprising a C-terminal extension sequence of RD ID (positions 241-244) with mutations to cysteines at those positions, as described herein. In another embodiment, a trimeric ligand comprises a first monomer, a second monomer, and a third monomer. In some embodiments, the monomers each comprise a first, second, and third variant CD27L protein. Each of the variant CD27L proteins can comprise a modification at one or more aminoDocket No. BREACH-001 / WOOl acid positions selected from the group consisting of Q149 and SI 17, as compared to wild-type CD27L. In some embodiments, new cysteine residues are introduced into the monomers, resulting in new disulfide bonds forming between individual monomers in the trimeric ligand. This increases stability and promotes a particular configuration of the monomers in the trimeric ligand. In some embodiments, residues are modified to create non-covalent associations, such as salt bridges, between monomers for similar purposes. In some embodiments, other modifications are included to strengthen the interaction between monomers. In some embodiments, the trimeric ligand comprises one or more monomers described in Table 4, below. Various combinations of individual monomers including specific mutations, including additional modifications that may create disulfide bonds, increase stability, or prevent undesired oligomers are provided in Table 4, below.CD27L MonomersIn another embodiment, a trimeric ligand comprises a first monomer, a second monomer, and a third monomer. In some embodiments, the monomers each comprise a first, second, and third variant CD40L protein. Each of the variant CD40L proteins can comprise a modification at one or more amino acid positions selected from the group consisting of Cl 78, Cl 94, A208, T211, S213, C218, G219, and S222, as compared to wild-type CD40L. In some embodiments, new cysteine residues are introduced into the monomers, resulting in new disulfide bonds forming between individual monomers in the trimeric ligand. This increases stability and promotes a particular configuration of the monomers in the trimeric ligand. In some embodiments, residues are modified to create non-covalent associations, such as salt bridges, between monomers for similar purposes. In some embodiments, other modifications are included to strengthen the interaction between monomers. In some embodiments, the trimeric ligand comprises one or more monomers described in Table 5, below. Various combinations of individual monomers including specific mutations, including additionalDocket No. BREACH-001 AVOOl modifications that may create disulfide bonds, increase stability, or prevent undesired oligomers are provided in Table 5, below.Table 5: Exemplary Trimeric Ligands based on Combinations of Mutated and Wild-Type CD40L MonomersIn another embodiment, a trimeric ligand comprises a first monomer, a second monomer, and a third monomer. In some embodiments, the monomers each comprise a first, second, and third variant TL1A protein. Each of the variant TL1 A proteins can comprise a modification at one or more amino acid positions selected from the group consisting of R96, D146, F148, C163, M197, C203, D242, and F244, as compared to wild-type TL1A. In some embodiments, new cysteine residues are introducedDocket No. BREACH-001 / WOOl into the monomers, resulting in new disulfide bonds forming between individual monomers in the trimeric ligand. This increases stability and promotes a particular configuration of the monomers in the trimeric ligand. In some embodiments, residues are modified to create non-covalent associations, such as salt bridges, between monomers for similar purposes. In some embodiments, other modifications are included to strengthen the interaction between monomers. In some embodiments, the trimeric ligand comprises one or more monomers described in Table 6, below. Various combinations of individual monomers including specific mutations, including additional modifications that may create disulfide bonds, increase stability, or prevent undesired oligomers are provided in Table 6, below.Docket No. BREACH-001 / WOOlTable 6: Exemplary Trimeric Ligands based on Combinations of Mutated and Wild-TypeTL1A MonomersIn another embodiment, a trimeric ligand comprises a first monomer, a second monomer, and a third monomer. In some embodiments, the monomers each comprise a first, second, and third variant LTB protein. Each of the variant LTB proteins can comprise a modification at one or more amino acid positions selected from the group consisting of LI 38, G181, and S204, as compared to wild-type LTB. In some embodiments, new cysteine residues are introduced into the monomers, resulting in new disulfide bonds forming between individual monomers in the trimeric ligand. This increases stability and promotes a particular configuration of the monomers in the trimeric ligand. In some embodiments, residues are modified to create non-covalent associations, such as salt bridges, between monomers for similar purposes. In some embodiments, other modifications are included to strengthen the interaction between monomers. In some embodiments, the trimeric ligand comprises one or more monomers described in Table 7, below. Various combinations of individual monomers including specific mutations, including additional modifications that may create disulfide bonds, increase stability, or prevent undesired oligomers, are provided in Table 7, below.Docket No. BREACH-001 / WOOlLTB MonomersIn another embodiment, a trimeric ligand comprises a heterotrimeric complex comprising a first monomer, a second monomer, and a third monomer. In some embodiments, the first monomer is LTA and the second and third monomers are LTB. In some embodiments, the first monomer is wildtype LTA. In other embodiments, the first monomer is a variant LTA protein comprising a modification at one or more amino acid positions selected from the group consisting of Ml 54 and LI 64, as compared to wild-type LTA. In some embodiments, the second and / or third monomers are a variant LTB protein. In such embodiments, the variant LTB protein can comprise a modification at one or more amino acid positions selected from the group consisting of R160, G191, A192, Y212, and S214, as compared to wild-type LTB. In some embodiments, new cysteine residues are introduced into the monomers, resulting in new disulfide bonds forming between individual monomers in the trimeric ligand. This increases stability and promotes a particular configuration of the monomers in the trimeric ligand. In some embodiments, residues are modified to create non-covalent associations, such as salt bridges, between monomers for similar purposes. In some embodiments, other modifications are included to strengthen the interaction between monomers. In some embodiments, the trimeric ligand comprises one or more monomers described in Table 8, below. Various combinations of individual monomers including specific mutations, including additional modifications that may create disulfide bonds, increase stability, or prevent undesired oligomers, are provided in Table 8, below.DocketNo. BREACH-001 / WOOlLTA and LTB MonomersIn another embodiment, a trimeric ligand comprises a first monomer, a second monomer, and a third monomer. In some embodiments, the monomers each comprise a first, second, and third wild- type or variant 4-1BBL protein. Each of the variant 4-1BBL proteins can comprise a modification at one or more amino acid positions selected from the group consisting of G80, D184, R193, and T241, as compared to wild-type 4-1BBL. In some embodiments, new cysteine residues are introduced into the monomers, resulting in new disulfide bonds forming between individual monomers in the trimeric ligand. This increases stability and promotes a particular configuration of the monomers in the trimeric ligand. In some embodiments, residues are modified to create non-covalent associations, such as salt bridges, between monomers for similar purposes. In some embodiments, other modifications are included to strengthen the interaction between monomers. In some embodiments, the trimeric ligand comprises one or more monomers described in Table 9, below. Various combinations of individual monomers including specific mutations, including additional modifications that may create disulfide bonds, increase stability, or prevent undesired oligomers, are provided in Table 9, below.Table 9: Exemplary Trimeric Ligands based on Combinations of Mutated and Wild-Type 4- 1BBL MonomersIn another embodiment, a trimeric ligand comprises a first monomer, a second monomer, and a third monomer. In some embodiments, the monomers each comprise a first, second, and third wild-Docket No. BREACH-001 / WOOl type or variant TRAIL protein. Each of the variant TRAIL proteins can comprise a modification at one or more amino acid positions selected from the group consisting of D203, C230, and S232, as compared to wild-type TRAIL. In some embodiments, new cysteine residues are introduced into the monomers, resulting in new disulfide bonds forming between individual monomers in the trimeric ligand. In some embodiments, one naturally occurring cysteine (C230) is mutated in one monomer to promote the formation of a disulfide bond between the two monomers where C230 is present. This increases stability and promotes a particular configuration of the monomers in the trimeric ligand. In some embodiments, residues are modified to create non-covalent associations, such as salt bridges, between monomers for similar purposes. In some embodiments, other modifications are included to strengthen the interaction between monomers. In some embodiments, the trimeric ligand comprises one or more monomers described in Table 10, below. Various combinations of individual monomers including specific mutations, including additional modifications that may create disulfide bonds, increase stability, or prevent undesired oligomers, are provided in Table 10, below.TRAIL MonomersAs shown in several of the Examples herein, the individual monomers of many of the trimeric ligands described in Tables 3-10 associate predominantly in a specific configuration, i.e., the trimeric ligand is polarized. Trimeric ligands according to the disclosure can employ modifications in individual monomers that promote a particular configuration of the trimeric ligand. For example, a trimeric ligand comprising monomers A, B, C will, due to new covalent and non-covalent associations between monomers, assemble such that A will interact with B and C at a specific interface, B will interact with A and C at a specific interface, and C will interact with A and B at a specific interface. This property (as may be referred to herein as “polarized”) may be leveraged to create new multifunctional and / or multispecific molecules, e.g., by fusing functional moieties to the amino and carboxyl termini of individual monomers. Because these monomers will associate in a particularDocket No. BREACH-001 / WOOl configuration, the number, location, and positioning of the functional moieties can be selected and specifically designed to create new therapeutic molecules with consistent expression, function, and potency.In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the trimeric ligands within a solution will comprise a particular configuration. For example, in a solution comprising monomers A, B, and C, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the trimeric ligands will comprise a trimer A-B-C, whereas others may comprise different undesired oligomers such as A-A-A, A-A-B, B-C-B, etc.In some embodiments, a trimeric ligand is coupled, e.g., covalently linked or fused, to one or more functional moieties. Such functional moieties can include but are not limited to: (i) tumor targeting moieties, e.g., a first tumor-targeting moiety, that binds to a cancer antigen; (ii) immune cell engagers, such as an NK cell engager, T cell engager, B cell engager, dendritic cell engager, or macrophagic cell engager; (iii) cytokine molecules; (iv) stromal modifying moieties; (v) checkpoint inhibitors; (vi) enzymes; and (vii) half-life extension moieties; as further described herein. Each of these moieties may be fused to individual monomers within the trimeric ligand by, e.g., a linker, such as a peptide linker. In some embodiments, a trimeric ligand can be multispecific, e g., by including two or more tumor targeting moieties. In any embodiment, the trimeric ligand can also be multifunctional, e.g., by including one or more of (ii), (iii), (iv), (v), (vi), and / or (vii).In some embodiments, a trimeric ligand is coupled to one or more of the following: Fab, Fv, scFv, single domain antibody, peptide, cytokine, or enzyme.In some embodiments, each trimeric ligand is coupled to a half-life extension moiety. The half-life extension moiety can be a human serum albumin binding protein, such as a single domain antibody or other type of binding protein. The half-life extension moiety can be a heavy chain constant region (e.g., an Fc region), e.g., a homodimeric or heterodimeric heavy chain constant region. The trimeric ligand can be coupled to the heavy chain, e.g., at the N- or C-terminus of the heavy chain constant region. The half-life extension moiety can be functionalized by having the Fc region bind to Fc receptors, thus activating immune response. This provides various beneficial properties such as half-life extension and immune engaging functions, e.g., antibody-dependent cellular cytotoxicity (ADCC,) antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).In other embodiments, the trimeric ligand is coupled, e.g., covalently linked or fused, to a constant domain of a Fab region. In some embodiments, the trimeric ligand is coupled, e.g., covalentlyDocket No. BREACH-001 / WOOl coupled or fused, to the N-terminus of the heavy chain variable constant region (e.g., a Fab CHI) and / or to the N-terminus of the light chain variable constant region (e.g., a Fab CL).In some embodiments, the trimeric ligand includes three monomer molecules, e.g., wherein two of the monomer molecules are coupled, e.g., covalently linked, to one another, and the third monomer molecule is non-covalently associated to the other two monomer molecules. In some embodiments, the multifunctional molecule comprises two, three or more trimeric ligands that are the same or different.In some embodiments, the trimeric ligand in the multifunctional molecule is a homotrimer, e.g., is composed of the same monomer molecules, or a heterotrimer, e.g., is composed of two or three different monomer molecules. In some embodiments, the trimeric ligand is a member of the tumor necrosis factor superfamily (TNFSF) or TNFSF-like members, or a combination of TNFSF- and TNFSF-like monomers.(b) Targeting Moieties, Tumor Targeting Moieties, and Antibody MoleculesIn certain embodiments, the multifunctional trimeric ligands disclosed herein include a targeting moiety which can preferentially bind to and target certain epitopes. A targeting moiety may be covalently joined to individual monomers within the trimeric ligand by a linker, such as a peptide linker, to either or both of the amino or carboxyl terminus. The targeting moiety can be chosen from an antibody molecule (e.g., an antigen binding domain as described herein), a receptor or a receptor fragment, a ligand or a ligand fragment, or a combination thereof.In some embodiments, the multifunctional trimeric ligands disclosed herein can include a tumor-targeting moiety. The terms “tumor” and “cancer” are used interchangeably herein and include all malignant and pre-malignant cancerous conditions. In some embodiments, the targeting moiety associates with, e g., binds to, a tumor cell (e.g., a molecule, e g., antigen, present on the surface of the tumor cell). In some embodiments, the targeting moiety binds to a stromal antigen such as an extracellular matrix antigen stromal cell antigen. In certain embodiments, the tumor targeting moiety targets, e.g., directs the multifunctional molecules disclosed herein to a cancer (e.g., a cancer or tumor cells). In some embodiments, the cancer is chosen from a hematological cancer, a solid cancer, a metastatic cancer, or a combination thereof.In some embodiments, a tumor-targeting moiety comprises an antibody molecule, a receptor molecule (e.g., a receptor, a receptor fragment or functional variant thereof), or a ligand molecule (e.g., a ligand, a ligand fragment or functional variant thereof) that binds to the cancer antigen. For example, the tumor-targeting moiety can bind to a cancer antigen present on a hematological cancer,Docket No. BREACH-001 / WOOl a solid tumor, a metastatic cancer, soft tissue tumor, metastatic lesion, or a combination thereof. In other embodiments, the cancer antigen is a tumor antigen or stromal antigen, or a hematological antigen. The tumor antigen or stromal antigen can be present on a fibrotic or desmoplastic solid tumor. For example, the tumor antigen or stromal antigen is present on a tumor, e.g., a tumor of a class typified by having one or more of: limited tumor perfusion, compressed blood vessels, or fibrotic tumor interstitium.In some embodiments, a tumor-targeting moiety binds to a solid tumor antigen or a stromal antigen. The solid tumor antigen or stromal antigen can be present on a solid tumor, or a metastatic lesion thereof. In some embodiments, the solid tumor is chosen from one or more of pancreatic (e.g., pancreatic adenocarcinoma), breast, colorectal, lung (e.g., small or non-small cell lung cancer), skin, ovarian, or liver cancer. In one embodiment, the solid tumor is a fibrotic or desmoplastic solid tumor. For example, the solid tumor antigen or stromal antigen can be present on a tumor, e.g., a tumor of a class typified by having one or more of: limited tumor perfusion, compressed blood vessels, or fibrotic tumor interstitium.In some embodiments, the tumor-targeting moiety comprises an antibody molecule, a receptor molecule (e.g., a receptor, a receptor fragment or functional variant thereof), or a ligand molecule (e.g., a ligand, a ligand fragment or functional variant thereof), or a combination thereof, that binds to the cancer antigen. For example, the tumor-targeting moiety can bind to a cancer antigen present on a hematological cancer, a solid tumor, a metastatic cancer, soft tissue tumor, metastatic lesion, or a combination thereof. In other embodiments, the cancer antigen is a tumor antigen or stromal antigen, or a hematological antigen. The tumor antigen or stromal antigen can be present on a fibrotic or desmoplastic solid tumor. For example, the tumor antigen or stromal antigen is present on a tumor, e.g., a tumor of a class typified by having one or more of: limited tumor perfusion, compressed blood vessels, or fibrotic tumor interstitium.Exemplary cancers that can be targeted include, but are not limited to the tumor, e.g., solid tumor, pancreatic (e.g., pancreatic adenocarcinoma), breast, colorectal, lung (e.g., small or non-small cell lung cancer), skin, ovarian, or liver cancer. The cancer can also be a hematological cancer including, but not limited to, B-cell or T cell malignancy, e.g., Hodgkin's lymphoma, Non-Hodgkin’s lymphoma (e.g., B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia), acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, and acute lymphocytic leukemia.Docket No. BREACH-001 / WOOlIn some embodiments, the cancer, e.g., solid tumor, antigen is chosen from: PD-L1, CD47, mesothelin, gangloside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), DLL3, Ron Kinase, c-Met, Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloride channel 2, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2 / neu, Telomerase, SAP-1, STEAP1, STEAP2, B7-H3, Survivin, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, TRP-1 / -2, MC1R, P-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, p53, Ras, TGF-B receptor, AFP, ETA, DLL3, PSA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, P-catenin, CDK4, CDC27, CD47, a actinin-4, TRPl / gp75, TRP2, gplOO, Melan-A / MARTl, gangliosides, WT1, EphA3, Epidermal growth factor receptor (EGFR), Claudin 18.2, CD20, MART -2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, RCC, RUH, RUI2, SAGE, TRG, TRP1, TSTA, Folate receptor alpha, Ll-CAM, CAIX, EGFRvIII, gpA33, GD3, GM2, VEGFR, Intergrins (Integrin alphaVbeta3, Integrin alpha5Betal), Carbohydrates (Le), IGF1R, EPHA3, TRAILR1, TRAILR2, or RANKL. In other embodiments, the cancer antigen is a stromal antigen can be chosen from fibroblast activating protease (FAP), TGF-beta, hyaluronic acid, collagen, e g., collagen IV, tenascin C, or tenascin W. In embodiments where the cancer antigen is a hematological antigen, the cancer antigen can be chosen from CD 19, CD33, CD47, CD 123, CD20, CD99, CD30, BCMA, CD38, CD22, SLAMF7, orNY-ESOl.In some embodiments of any of the multispecific or multifunctional molecules disclosed herein, the tumor-targeting moiety is chosen from an antibody molecule to a cancer antigen chosen from mesothelin, PD-L1, HER3, IGF1R, FAP, CD47 or CD123. For example, the tumor-targeting moiety can include an antibody molecule (e.g., Fab or scFv) that binds to mesothelin or PD-L1. In some embodiments, the tumor-targeting moiety binds to PD-L1 and inhibits an interaction of PD-L1 with PD1. In other embodiments, the tumor-targeting moiety binds to PD-L1 and does not inhibit an interaction of PD-L1 with PD1.In one embodiment, the tumor-targeting moiety includes an antibody molecule (e.g., Fab or scFv) that binds to PD-L1.In some embodiments, the tumor targeting moiety can comprise a tumor targeting peptide, such as the iRGD peptide.In one embodiment, a tumor-targeting moiety binds to an antigen, e g., an immune effector cell, a tumor antigen or a stromal antigen. In some embodiments, the antigen is, e.g., a mammalian, e.g., a human, antigen. In other embodiments, the antibody molecule binds to an immune cell antigen,Docket No. BREACH-001 / WOOl e.g., a mammalian, e.g., a human, immune cell antigen. For example, the antibody molecule binds specifically to an epitope, e.g., linear or conformational epitope, on the cancer antigen or the immune cell antigen.In an embodiment, a tumor-targeting moiety is a monospecific antibody molecule and binds a single epitope. E.g., a monospecific antibody molecule having a plurality of immunoglobulin variable domain sequences, each of which binds the same epitope.In an embodiment, a tumor-targeting moiety comprises a diabody, and a single-chain molecule, as well as an antigen-binding fragment of an antibody (e.g., Fab, F(ab’)2, and Fv). For example, a tumor-targeting moiety can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, the VH and VL are linked to the same monomer, e.g., a VH fused to the amino terminus and a VL fused to the carboxyl terminus of the monomer, as shown in FIG. 2B, to create the targeting moiety. In other embodiments, the VH and VL are linked to separate monomers, e.g., a VH fused to either terminus of a first monomer and a VL fused to either terminus of a second monomer, such that the VH and VL interact to form an antigen binding domain, as shown in FIG. 2B, to create the targeting moiety. As also shown below in the Examples, a VH and VL interacting to form a functional antigen-binding fragment across two monomers can also help promote the association of the monomers into a particular configuration. In some embodiments, a cysteine residue may be added to the VH and VL, thereby resulting in a disulfide bond between the two.In an embodiment a tumor targeting moiety comprises an antibody molecule comprising a heavy chain and a light chain (which may be referred to herein as a half antibody). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen binding sites. In various embodiments, antibody molecules can comprise Fab, Fab’, F(ab’)2, Fc, Fd, Fd’, Fv, single chain antibodies (scFv for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen or receptor.An antibody molecule can also be a human, humanized, CDR-grafted, or in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or lgG4. The antibody can also have a light chain chosen from, e.g., kappa or lambda. The term “immunoglobulin” (Ig) is used interchangeably with the term antibody herein.Docket No. BREACH-001 / WOOlExamples of antigen-binding fragments of a tumor-targeting moiety include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv), see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883); (viii) a single domain antibody. These antibody fragments may be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.Tumor targeting moieties can include intact antibody molecules as well as functional fragments thereof. Constant regions of the antibody molecules can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).Tumor targeting moieties can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. For example, humanized mouse antibodies can be engineered as single domain antibodies, and certain human antibodies of the IGVH3 type closely resemble camelid VHH domains. Some IGVH3 subtypes naturally express without a light chain, and transgenic mice can be developed to express such heavy-chain-only antibodies, making these human-derived antibodies (discovered in mice) practical alternatives to camelid sources.According to another aspect of the disclosure, a single domain antibody is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678, for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHHDocket No. BREACH-001 / WOOl molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the disclosure.In an antibody molecule, the VH and VL regions can be subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR), interspersed with regions that are more conserved, termed "framework regions" (FR or FW).The extent of the framework region and CDRs has been precisely defined by a number of methods (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular’s AbM antibody modeling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains, in: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer- Verlag, Heidelberg).The terms “complementarity determining region,” and “CDR,” as used herein refer to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3).The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme). As used herein, the CDRs defined according to the “Chothia” number scheme are also sometimes referred to as “hypervariable loops.”For example, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).Each VH and VL typically includes three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.Docket No. BREACH-001 / WOOlA trimeric ligand with various tumor-targeting moieties can be recombinantly produced, e.g., produced by phage display or by combinatorial methods. Phage display and combinatorial methods for generating antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al., International Publication No. WO 92 / 18619; Dower et al. International Publication No. WO 91 / 17271; Winter et al. International Publication WO 92 / 20791; Markland et al. International Publication No. WO 92 / 15679; Breitling et al. International Publication WO 93 / 01288; McCafferty et al. International Publication No. WO 92 / 01047; Garrard et al. International Publication No. WO 92 / 09690; Ladner et al. International Publication No. WO 90 / 02809; Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246: 1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) 5 PNAS 89:3576-3580; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).In one embodiment, the tumor targeting moiety is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods of producing rodent antibodies are known in the art.Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91 / 00906, Kucherlapati et al. PCT publication WO 91 / 10741; Lunberg et al. International Application WO 92 / 03918; Kay et al. International Application 92 / 03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L.L. et al. 1994 Nature Genet. 20 7: 13-21; Morrison, S.L. et al. 1994 Proc. Natl. Acad. Sci. USA 81 :6851-6855; Bruggeman et al. 1993 Eur J Immunol 1:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21: 1323-1326).An “effectively human” protein is a protein that does substantially not evoke a neutralizing antibody response, e.g., the human anti-murine antibody (HAMA) response. HAMA can be problematic in several circumstances, e.g., if the antibody molecule is administered repeatedly, e.g., in treatment of a chronic or recurrent disease condition. A HAMA response can make repeatedDocket No. BREACH-001 / WOOl antibody administration potentially ineffective because of an increased antibody clearance from the serum (see, e.g., Saleh et al. Cancer Immunol. Immunother., 32: 180-190 (1990)) and also because of potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication PCT7US86 / 02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240: 1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80: 1553-1559).A tumor-targeting moiety comprising a humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDRs (of heavy and or light immunoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding to the antigen. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDRs is called the “donor” and the immunoglobulin providing the framework is called the “acceptor”. In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A "consensus framework" refers to the framework region in the consensus immunoglobulin sequence.An antibody molecule can be humanized by methods known in the art (see e.g., Morrison, S. L., 1985, Science 229: 1202-1207, by Oi et al., 1986, Bio Techniques 4:214, and by Queen et al. USDocket No. BREACH-001 / WOOl5,585,089, US 5,693,761 and US 5,693,762, the contents of all of which are hereby incorporated by reference).Humanized or CDR-grafted antibody molecules can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CD Rs of an immunoglobulin chain can be replaced. See e.g., U.S. Patent 5,225,539; Jones et al. 1986 Nature 321 :552-525; Verhoeyan et al. 1988 Science 239: 1534; Beidler et al. 1988 J. Immunol. 141 :4053-4060; Winter US 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present disclosure (UK Patent Application GB 2188638A, filed on March 26, 1987; Winter US 5,225,539), the contents of which are expressly incorporated by reference.Also within the scope of the disclosure are tumor-targeting moieties comprising humanized antibody molecules in which specific amino acids have been substituted, deleted or added. Criteria for selecting amino acids from the donor are described in US 5,585,089, e.g., columns 12-16 of US 5,585,089, e.g., columns 12-16 of US 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 Al, published on December 23, 1992.The tumor targeting moiety can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target protein.In yet other embodiments, an antibody molecule has a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgGl, TgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgGl, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and / or complement function). In one embodiment the antibody has: effector function; and can fix complement. In other embodiments the antibody does not recruit effector cells or fix complement. In another embodiment, the antibody has reduced or no ability to bind an FcDocket No. BREACH-001 / WOOl receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.Methods for altering an antibody constant region are known in the art. Antibodies with altered function, e.g. altered affinity for an effector ligand, such as FcR on a cell, or the Cl component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388,151 Al, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents of all of which are hereby incorporated by reference). Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.An antibody molecule can be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a “derivatized” antibody molecule is one that has been modified. Methods of derivatization include but are not limited to the addition of a fluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinity ligand such as biotin. Accordingly, the antibody molecules as disclosed herein may include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag). Antibody molecules may also be fused to trimeric ligands, as described further herein.One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bi specific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce 30 Chemical Company, Rockford, Ill.In an embodiment a tumor-targeting moiety is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domains sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment the first and second epitopes are on the same antigen, e.g., the same proteinDocket No. BREACH-001 / WOOl(or subunit of a multimeric protein). In an embodiment the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In an embodiment the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment a multispecific antibody molecule comprises a third, fourth or fifth immunoglobulin variable domain. In an embodiment, a multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.It should be noted that antibody molecules as described herein can be employed as both targeting (e.g., any antigen) or tumor-targeting (e.g., cancer antigen) moieties. Accordingly, any disclosure herein referencing a tumor-targeting moiety may equally be applied to target other antigens, such as checkpoint inhibitors (e.g., PD-1, PD-L1, CTLA-4) and immune cell engagers, as further described below. Various embodiments are considered to be within the scope of the disclosure.(c) Cytokine MoleculesIn certain embodiments, the multifunctional trimeric molecules disclosed herein can further include a cytokine molecule other than a TNFSF or TNF-like family member. Such cytokine molecules can be covalently attached to individual monomers of the trimeric molecule by a linker (e.g., a peptide linker) at either or both of the amino and carboxyl termini.Cytokines are proteinaceous signaling compounds that are mediators of the immune response. They control many different cellular functions including proliferation, differentiation and cell survival / apoptosis; cytokines are also involved in several pathophysiological processes including viral infections and autoimmune diseases. Cytokines are synthesized under various stimuli by a variety of cells of both the innate (monocytes, macrophages, dendritic cells) and adaptive (T- and B-cells) immune systems. Cytokines can be classified into two groups: pro- and anti-inflammatory. Pro- inflammatory cytokines, including IFNgamma, IL-I, IL-6 and TNF alpha, are predominantly derived from the innate immune cells and Thl cells. Anti-inflammatory cytokines, including IL-10, IL-4, IL- 13 and IL-5, are synthesized from Th2 immune cells.The present disclosure provides, inter alia, specific and multi-specific (e.g., bi-, tri-, quadspecific) and / or multifunctional proteins, that include, e g., are engineered to contain, one or more cytokine molecules, e.g., immunomodulatory (e.g., proinflammatory) cytokines and variants, e.g., functional variants, thereof. Accordingly, in some embodiments, the cytokine molecule is an interleukin or a variant, e.g., a functional variant thereof. In some embodiments the interleukin is a proinflammatory interleukin. In some embodiments the interleukin is chosen from interleukin-2 (IL- 2), interleukin- 12 (IL-12), interleukin-15 (IL-15), interleukin- 18 (IL-18), interleukin-21 (IL-21), orDocket No. BREACH-001 / WOOl interferon gamma. In some embodiments, the cytokine molecule is a proinflammatory cytokine. In some embodiments the interleukin is an anti-inflammatory interleukin. In some embodiments the interleukin is chosen from interl eulin- 10 (IL-10), interleulin-4 (IL-4), interleulin-13 (IL-13), interleulin-5 (IL-5).In some embodiments, the multifunctional molecules disclosed herein include a cytokine molecule. In embodiments, the cytokine molecule includes a full length, a fragment or a variant of a cytokine; a cytokine receptor domain, e.g., a cytokine receptor dimerizing domain; or an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) to a cytokine receptor.In some embodiments the cytokine molecule is chosen from IL-2, IL-12, IL-15, IL-18, IL-21, or interferon gamma, or a fragment or variant thereof, or a combination of any of the aforesaid cytokines. The cytokine molecule can be a monomer molecule or a dimer. In embodiments, the cytokine molecule can further include a cytokine receptor dimerizing domain.In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) to a cytokine receptor chosen from an IL-15Ra or IL- 21R.In other embodiments, the cytokine molecule is chosen from IL-10, IL-4, IL-13, IL-5.(d) Immune Cell EngagersIn certain embodiments, the multifunctional trimer molecules disclosed herein include an immune cell engager, i.e., an immune cell engaging moiety. Like other functional units disclosed herein, immune cell engagers can be fused to a trimeric ligand to create multifunctional and / or multispecific molecules.The immune cell engagers of the multifunctional molecules disclosed herein can mediate binding to, and / or activation of, an immune cell, e.g., an immune effector cell. In some embodiments, the immune cell is chosen from an NK cell, a B cell, a T cell, a dendritic cell, a macrophage cell engager, a toll cell receptor, or a combination thereof. In some embodiments, the immune cell engager is chosen from one, two, three, four, or more of an NK cell engager, a B cell engager, a T cell engager, a dendritic cell engager, a macrophage cell engager, or a combination thereof. The immune cell engager can be an agonist of the immune system. In some embodiments, the immune cell engager can be an antibody molecule (e.g., a targeting moiety which targets immune cells), a ligand molecule (e.g., a ligand that further comprises an immunoglobulin constant region, e.g., an Fc region), a small molecule, or a nucleotide molecule.Docket No. BREACH-001 / WOOlIn one embodiment, an immune cell engager is a dendritic cell engager. In one embodiment, the dendritic cell engager is an antibody (e.g., a single domain antibody) that targets CLEC9A. The dendritic cell engager specifically binds to CLEC9A, a receptor expressed on a subset of dendritic cells known as cDCl. When combined in a multispecific antibody format, the immune cell engager also includes an anti-CD3 domain that interacts with T cells. By simultaneously binding CLEC9A on cDCl and CD3 on T cells, this engager brings cDCl cells into close proximity with T cells. This interaction primes the T cells, enabling them to establish long-term memory. As a result, the trained T cells are able to recognize and kill tumor cells even after initial engagement, offering a sustained anti-tumor effect by equipping the immune system to target these cells over time.In some embodiments, the immune cell engager comprises a T cell engager that mediates binding to and activation of, a T cell. In some embodiments, the immune cell engager comprises a T cell engager that mediates binding to but not activation of, a T cell.In other embodiments, the immune cell engager mediates binding to, or activation of, or both of, one or more of a B cell, T cell, a macrophage, and / or a dendritic cell.In other embodiments, the T cell engager is an agonist of CD3, TCRa, TCR[3, TCRy, TCRq, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In other embodiments, the T cell engager binds to, but does not CD3, TCRa, TCR0, TCRy, TCRq. ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226In some embodiments, the immune cell engager comprises a B cell, macrophage, and / or dendritic cell engager chosen from one or more of CD40 ligand (CD40L) or a CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule to 0X40; an 0X40 ligand (OX40L); an agonist of a Toll-like receptor (e g., a TLR4, e g., a constitutively active TLR4 (caTLR4) or a TLR9 agonist); a 41BB; a CD2 agonist; a CD47; or a STING agonist, or a combination thereof.In some embodiments, the B cell engager is a CD40L, an OX40L, or a CD70 ligand, or an antibody molecule that binds to 0X40, CD40 or CD70.In other embodiments, the macrophage cell engager is a CD2 agonist; a CD40L; an OX40L; an antibody molecule that binds to 0X40, CD40 or CD70; an agonist of a Toll-like receptor (TLR)(e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4) or a TLR9 agonist); CD47; or a STING agonist.In yet other embodiments, the dendritic cell engager is a CD2 agonist, an 0X40 antibody, an OX40L, a CLEC9A antibody, a 41BB agonist, a Toll-like receptor agonist or a fragment thereof (e.g.,Docket No. BREACH-001 / WOOl a TLR4, e.g., a constitutively active TLR4 (caTLR4)), CD47 agonist or a STING agonist. For example, the STING agonist can include a cyclic dinucleotide, e.g., a cyclic di-GMP (cdGMP), a cyclic di-AMP (cdAMP), or a combination thereof, optionally with 2', 5' or 3 ',5' phosphate linkages. The STING agonist can be covalently coupled to the multispecific or multifunctional molecule, e.g., by art known techniques.NK Cell EngagersIn some embodiments, the immune cell engaging moiety is a Natural Killer cell engager. Natural Killer (NK) cells recognize and destroy tumors and virus-infected cells in an antibodyindependent manner. The regulation of NK cells is mediated by activating and inhibiting receptors on the NK cell surface. One family of activating receptors is the natural cytotoxicity receptors (NCRs) which include NKp30, NKp44 and NKp46. The NCRs initiate tumor targeting by recognition of heparan sulfate on cancer cells. NKG2D is a receptor that provides both stimulatory and costimulatory innate immune responses on activated killer (NK) cells, leading to cytotoxic activity. DNAM1 is a receptor involved in intercellular adhesion, lymphocyte signaling, cytotoxicity and lymphokine secretion mediated by cytotoxic T lymphocyte (CTL) and NK cell. DAP10 (also known as HCST) is a transmembrane adapter protein which associates with KLRK1 to form an activation receptor KLRK1-HCST in lymphoid and myeloid cells; this receptor plays a major role in triggering cytotoxicity against target cells expressing cell surface ligands such as MHC class I chain-related MICA and MICB, and U(optionally Ll)6-binding proteins (ULBPs); it KLRK1-HCST receptor plays a role in immune surveillance against tumors and is required for cytolysis of tumor cells; indeed, melanoma cells that do not express KLRK1 ligands escape from immune surveillance mediated by NK cells. CD16 is a receptor for the Fc region of IgG, which binds complexed or aggregated IgG and also monomer IgG and thereby mediates antibody-dependent cellular cytotoxicity (ADCC) and other antibody-dependent responses, such as phagocytosis.In some embodiments, the NK cell engager is a viral hemagglutinin (HA), HA is a glycoprotein found on the surface of influenza viruses. It is responsible for binding the virus to cells with sialic acid on the membranes, such as cells in the upper respiratory tract or erythrocytes. HA has at least 18 different antigens. These subtypes are named Hl through Hl 8. NCRs can recognize viral proteins. NKp46 has been shown to be able to interact with the HA of influenza and the HA-NA of Paramyxovirus, including Sendai virus and Newcastle disease virus. Besides NKp46, NKp44 can also functionally interact with HA of different influenza subtypes.Docket No. BREACH-001 / WOOlThe present disclosure provides, inter alia, multi-specific (e.g., bi-, tri-, quad-specific) and / or multifunctional proteins based on TNFSF ligands, which may be engineered to contain one or more NK cell engagers that mediate binding to and / or activation of an NK cell. Accordingly, in some embodiments, the NK cell engager mediates binding to and activation of an NK cell; in other embodiments, the NK cell engager mediates binding without activation. Exemplary NK cell engagers may be selected from antibody molecules, such as antigen binding domains or ligands that bind to (e.g., activate): NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160. In some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen binding domain that binds specifically to NKp30 or NKp46.In other embodiments, the NK cell engager is a ligand of NKp44 or NKp46, which is a viral HA. Viral hemagglutinins (HA) are glyco proteins which are on the surface of viruses. HA proteins allow viruses to bind to the membrane of cells via sialic acid sugar moieties which contributes to the fusion of viral membranes with the cell membranes (see e.g., Eur J Immunol. 2001 Sep;31(9):2680-9 “Recognition of viral hemagglutinins by NKp44 but not by NKp30” and Nature 2001 Feb 22;409(6823): 1055-60 “Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells”.In other embodiments, the NK cell engager is a ligand of NKG2D chosen from MICA, MICB, or ULBP1.In other embodiments, the NK cell engager is a ligand of DNAM1 chosen from NECTIN2 or NECL5.In yet other embodiments, the NK cell engager is a ligand of DAP10, which is an adapter for NKG2D (see e.g., Proc Natl Acad Sci USA 2005 May 24; 102(21): 7641-7646; and Blood, September 2011 Volume 118, Number 11.In other embodiments, the NK cell engager is a ligand of CD16, which is a CD16a / b ligand, e.g., a CD16a / b ligand further comprising an antibody Fc region (see e.g., Front Immunol. 2013; 4: 76 discusses how antibodies use the Fc to trigger NK cells through CD 16. Such ligands can include single domain antibodies targeting CD 16.In other embodiments the NK cell engager is a ligand, optionally, the ligand further comprises an immunoglobulin constant region, e.g., an Fc region. For example, the ligand of NKp44 or NKp46Docket No. BREACH-001 / WOOl is a viral HA; the ligand of DAP10 is a coreceptor for NK.G2D; the ligand of CD16 is a CD16a / b ligand, e.g., a CD16a / b ligand further comprising an antibody Fc region.B Cell, Macrophage, & Dendritic Cell EngagersIn some embodiments, the immune cell engaging moiety is a B cell engager. Broadly, B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system by secreting antibodies. Additionally, B cells present antigen (they are also classified as professional antigen-presenting cells (APCs)) and secrete cytokines. Macrophages are a type of white blood cell that engulfs and digests cellular debris, foreign substances, microbes, and cancer cells via phagocytosis. Besides phagocytosis, they play important roles in nonspecific defense (innate immunity) and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. For example, they are important as antigen presenters to T cells. Beyond increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can decrease immune reactions through the release of cytokines. Dendritic cells (DCs) are antigen- presenting cells that function in processing antigen material and present it on the cell surface to the T cells of the immune system.The present disclosure provides, inter alia, multi-specific and / or multifunctional trimeric ligands, that may include, e.g., are engineered to contain, one or more B cell, macrophage, and / or dendritic cell engagers that mediate binding to and / or activation of a B cell, macrophage, and / or dendritic cell.Accordingly, in some embodiments, the immune cell engager comprises a B cell, macrophage, and / or dendritic cell engager chosen from one or more of CD40 ligand (CD40L) or a CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule to 0X40; an 0X40 ligand (OX40L); an agonist of a Toll-like receptor (e.g., as described herein, e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4), or a TLR9 agonists); a 41B8; a CD2; a CD47; or a STING agonist, or a combination thereof.In some embodiments, the B cell engager is a CD40L, an OX40L, or a CD70 ligand, or an antibody molecule that binds to 0X40, CD40 or CD70, e.g., also described herein as TNFSF family members.In some embodiments, the macrophage engager is a CD2 agonist. In some embodiments, the macrophage engager is an antigen binding domain that binds to: CD40L or antigen binding domain or ligand that binds CD40, a Toll like receptor {TLR) agonist (e.g., as described herein), e.g., a TLR9Docket No. BREACH-001 / WOOl or TLR4 (e.g., caTLR4 (constitutively active TLR4), CD47, or a STING agonist. In some embodiments, the STING agonist is a cyclic dinucleotide, e.g., cyclic di-GMP (cdGMP) or cyclic diAMP (cdAMP). In some embodiments, the STING agonist is biotinylated.In some embodiments, the dendritic cell engager is a CD2 agonist. In some embodiments, the dendritic cell engager is a ligand, a receptor agonist, or an antibody molecule that binds to one or more of: OX40L, 41BB, a TLR agonist (e.g., as described herein) (e.g., TLR9 agonist, TLR4 (e.g., caTLR4 (constitutively active TLR4)), CD47, or and a STING agonist. In some embodiments, the STING agonist is a cyclic dinucleotide, e.g., cyclic di-GMP (cdGMP) or cyclic di-AMP (cdAMP). In some embodiments, the STING agonist is biotinylated.In other embodiments, the immune cell engager mediates binding to, or activation of, one or more of a B cell, a macrophage, and / or a dendritic cell. Exemplary B cell, macrophage, and / or dendritic cell engagers can be chosen from one or more of CD40 ligand (CD40L) or a CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule to 0X40; an 0X40 ligand (OX40L); a Toll-like receptor agonist (e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4) or a TLR9 agonist); a 41BB agonist; a CD2; a CD47; or a STING agonist, or a combination thereof.In some embodiments, the B cell engager is chosen from one or more of a CD40L, an OX40L, or a CD70 ligand, or an antibody molecule that binds to 0X40, CD40 or CD70. In other embodiments, the macrophage cell engager is chosen from one or more of a CD2 agonist; a CD40L; an OX40L; an antibody molecule that binds to 0X40, CD40 or CD70; a Toll like receptor agonist or a fragment thereof (e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4)); a CD47 agonist; or a STING agonist.In other embodiments, the dendritic cell engager is chosen from one or more of a CD2 agonist, an 0X40 antibody, an OX40L, 41BB agonist, a Toll-like receptor agonist or a fragment thereof (e g., a TLR4, e.g., a constitutively active TLR4 (caTLR4)), CD47 agonist, or a STING agonist.In yet other embodiments, the STING agonist comprises a cyclic dinucleotide, e.g., a cyclic di-GMP (cdGMP), a cyclic di-AMP (cdAMP), or a combination thereof, optionally with 2', 5' or 3', 5' phosphate linkages.Toll Cell ReceptorsIn some embodiments, the immune cell engaging moiety is a toll cell receptor engaging moiety. Toll-Like Receptors (TLRs) are evolutionarily conserved receptors are homologues of the Drosophila Toll protein, and recognize highly conserved structural motifs known as pathogen associated microbial patterns (PAMPs), which are exclusively expressed by microbial pathogens, orDocket No. BREACH-001 / WOOl danger-associated molecular patterns (DAMPs) that are endogenous molecules released from necrotic or dying cells. P AMPs include various bacterial cell wall components such as lipopolysaccharide (LPS), peptidoglycan (PGN) and lipopeptides, as well as flagellin, bacterial DNA and viral doublestranded RNA. DAMPs include intracellular proteins such as heat shock proteins as well as protein fragments from the extracellular matrix. Stimulation of TLRs by the corresponding PAMPs or DAMPs initiates signaling cascades leading to the activation of transcription factors, such as AP-I, NF-KB and interferon regulatory factors (IRFs). Signaling byTLRs results in a variety of cellular responses, including the production of interferons (IFNs), pro- inflammatory cytokines and effector cytokines that direct the adaptive immune response. TLRs are implicated in a number of inflammatory and immune disorders and play a role in cancer (Rakoff- Nahoum S. & Medzhitov R., 2009. Toll-like receptors and cancer. Nat Revs Cancer 9:57- 63.)TLRs are type I transmembrane proteins characterized by an extracellular domain containing leucine-rich repeats (LRRs) and a cytoplasmic tail that contains a conserved region called the Toll / IL- 1 receptor (TER) domain. Ten human and twelve murine TLRs have been characterized, TLR1 to TLR10 in humans, and TLR1 to TLR9, TLR1 1, TLR12 and TLR13 in mice, the homolog of TLR10 being a pseudogene. TLR2 is essential for the recognition of a variety of PAMPs from Gram-positive bacteria, including bacterial lipoproteins, lipomannans and lipoteichoic acids. TLR3 is implicated in virus-derived double-stranded RNA. TLR4 is predominantly activated by lipopolysaccharide. TLR5 detects bacterial flagellin and TLR9 is required for response to unmethylated CpG DNA. Finally, TLR7 and TLR8 recognize small synthetic antiviral molecules, and single-stranded RNA was reported to be their natural ligand. TLR1 1 has been reported to recognize uropathogenic E.coli and a profilin- like protein from Toxoplasma gondii. The repertoire of specificities of the TLRs is apparently extended by the ability of TLRs to heterodimerize with one another. For example, dimers of TLR2 and TLR6 are required for responses to diacylated lipoproteins while TLR2 and TLR1 interact to recognize triacylated lipoproteins. Specificities of the TLRs are also influenced by various adapter and accessory molecules, such as MD-2 and CD 14 that form a complex with TLR4 in response to LPS.TLR signaling consists of at least two distinct pathways: a MyD88-dependent pathway that leads to the production of inflammatory cytokines, and a MyD88-independent pathway associated with the stimulation of IFN- and the maturation of dendritic cells. The MyD88- dependent pathway is common to all TLRs, except TLR3 (Adachi O. et al., 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity. 9(1): 143-50). Upon activation byDocket No. BREACH-001 / WOOlPAMPs or DAMPs, TLRs hetero- or homodimerize inducing the recruitment of adaptor proteins via the cytoplasmic TIR domain. Individual TLRs induce different signaling responses by usage of the different adaptor molecules. TLR4 and TLR2 signaling requires the adaptor TIRAP / Mal, which is involved in the MyD88-dependent pathway. TLR3 triggers the production of IFN-0 in response to double-stranded RNA, in a MyD88- independent manner, through the adaptor TRJF / TICAM-1. TRAM / TIC AM-2 is another adaptor molecule involved in the MyD88-independent pathway which function is restricted to the TLR4 pathway. TLR3, TLR7, TLR8 and TLR9 recognize viral nucleic acids and induce type I IFNs. The signaling mechanisms leading to the induction of type I IFNs differ depending on the TLR activated. They involve the interferon regulatory factors, IRFs, a family of transcription factors known to play a critical role in antiviral defense, cell growth and immune regulation. Three IRFs (IRF3, IRF5 and IRF7) function as direct transducers of virus-mediated TLR signaling. TLR3 and TLR4 activate IRF3 and IRF7, while TLR7 and TLR8 activate IRF5 and IRF7 (Doyle S. et al., 2002. IRF3 mediates a TLR3 / TLR4-specific antiviral gene program. Immunity. 17(3):251- 63). Furthermore, type I IFN production stimulated by TLR9 ligand CpG-A has been shown to be mediated by PI(3)K and mTOR (Costa-Mattioli M. & Sonenberg N. 2008. RAPping production of type I interferon in pDCs through mTOR. Nature Immunol. 9: 1097-1099).TLR9 recognizes unmethylated CpG sequences in DNA molecules. CpG sites are relatively rare (-1%) on vertebrate genomes in comparison to bacterial genomes or viral DNA. TLR9 is expressed by numerous cells of the immune system such as B lymphocytes, monocytes, natural killer (NK) cells, and plasmacytoid dendritic cells. TLR9 is expressed intracellularly, within the endosomal compartments and functions to alert the immune system of viral and bacterial infections by binding to DNA rich in CpG motifs. TLR9 signals leads to activation of the cells initiating pro-inflammatory reactions that result in the production of cytokines such as type-I interferon and IL-12.A TLR agonist can agonize one or more TLR, e.g., one or more of human TLR- 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, an adjunctive agent described herein is a TLR agonist. In some embodiments, the TLR agonist specifically agonizes human TLR-9. In some embodiments, the TLR- 9 agonist is a CpG moiety. As used herein, a CpG moiety, is a linear dinucleotide having the sequence: 5' — C — phosphate — G — 3', that is, cytosine and guanine separated by only one phosphate.In some embodiments, the CpG moiety comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more CpG dinucleotides. In some embodiments, the CpG moiety consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 CpG dinucleotides. In some embodiments, the CpGDocket No. BREACH-001 / WOOl moiety has 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 10- 20, 10-30, 10-40, or 10-50 CpG dinucleotides.In some embodiments, the TLR-9 agonist is a synthetic ODN (oligodeoxynucleotides). CpG ODNs are short synthetic single-stranded DNA molecules containing unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs). CpG ODNs possess a partially or completely phosphorothioated (PS) backbone, as opposed to the natural phosphodi ester (PO) backbone found in genomic bacterial DNA. There are three major classes of CpG ODNs: classes A, B and C, which differ in their immunostimulatory activities. CpG-A ODNs are characterized by a PO central CpG-containing palindromic motif and a PS-modified 3 ' poly-G string. They induce high IFN- a production from pDCs but are weak stimulators of TLR9-dependent NF-KB signaling and pro- inflammatory cytokine (e.g. IL-6) production. CpG-B ODNs contain a full PS backbone with one or more CpG dinucleotides. They strongly activate B cells and TLR9- dependent NF- B signaling but weakly stimulate IFN-a secretion. CpG-C ODNs combine features of both classes A and B. They contain a complete PS backbone and a CpG-containing palindromic motif. C-Class CpG ODNs induce strong IFN-a production from pDC as well as B cell stimulation.(e) Other functional moietiesIn some embodiments, the multispecific or multifunctional molecule can include a stromal modifying moiety that causes one or more of: decreases the level or production of a stromal or extracellular matrix (ECM) component; decreases tumor fibrosis; increases interstitial tumor transport; improves tumor perfusion; expands the tumor microvasculature; decreases interstitial fluid pressure (IFP) in a tumor; or decreases or enhances penetration or diffusion of an agent, e.g., a cancer therapeutic or a cellular therapy, into a tumor or tumor vasculature. For example, the stromal or ECM component decreased is chosen from a glycosaminoglycan or an extracellular protein, or a combination thereof. The glycosaminoglycan can be chosen from hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparan sulfate, heparin, entactin, tenascin, aggrecan or keratin sulfate. Exemplary extracellular proteins include, but are not limited to, collagen, laminin, elastin, fibrinogen, fibronectin, or vitronectin.In some embodiments, the multispecific or multifunctional molecule includes a stromal modifying moiety that comprises an enzyme molecule that degrades a tumor stroma or extracellular matrix (ECM). The enzyme molecule can be chosen from a hyaluronidase molecule, a collagenase molecule, a chondroitinase molecule, a matrix metalloproteinase molecule (e.g., macrophage metalloelastase), or a variant (e.g., a fragment) of any of the aforesaid.Docket No. BREACH-001 / WOOlIn some embodiments, the stromal modifying moiety decreases the level or production of hyaluronic acid. For example, the stromal modifying moiety comprises a hyaluronan degrading enzyme, an agent that inhibits hyaluronan synthesis, or an antibody molecule against hyaluronic acid.In yet other embodiments, the hyaluronan degrading enzyme is a hyaluronidase molecule or a variant (e.g., fragment thereof) thereof. The hyaluronan degrading enzyme can be active in neutral or acidic pH, e.g., pH of about 4-5. In some embodiments, the hyaluronidase molecule is a mammalian hyaluronidase molecule, e.g., a recombinant human hyaluronidase molecule, or a variant thereof (e.g., a truncated form thereof). For example, the hyaluronidase molecule can be chosen from HYAL1, HYAL2, or PH-20 / SPAM1, or a variant thereof (e.g., a truncated form thereof). In yet other embodiments, the truncated form lacks a C-terminal glycosylphosphatidylinositol (GPI) attachment site or a portion of the GPI attachment site.In yet other embodiments, the hyaluronidase molecule is glycosylated, e.g., comprises at least one N-linked glycan.In some embodiments, each trimeric ligand is coupled to a half-life extension moiety. The half-life extension moiety can be a heavy chain constant region (e.g., an Fc region), e.g., a homodimeric or heterodimeric heavy chain constant region. The trimeric ligand can be coupled to the heavy chain, e.g., at the N- or C-terminus of the heavy chain constant region. In some embodiments, the half-life extension moiety can be functionalized by having the Fc region bind to Fc receptors, thus activating immune response and providing beneficial properties such as half-life extension and immune-engaging functions (e.g., antibody-dependent cellular cytotoxicity (ADCC,) antibodydependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC)). In other embodiments, the trimeric ligand is coupled, e.g., covalently linked or fused, to a constant domain of a Fab region. For example, the trimeric ligand can be coupled, e.g., covalently coupled or fused, to the N-terminus of the heavy chain variable constant region (e g., a Fab CHI) and / or to the N-terminus of the light chain variable constant region (e.g., a Fab CL). In other embodiments, the half-life extension moiety can be a human serum albumin (HSA) binder, such as an anti-HSA antibody, anti- HSA scFv, anti-HSA single domain antibody (VHH, nanobody), or another type of HSA binding moiety.(f) Exemplary Multispecific and / or Multifunctional MoleculesEmbodiments of the disclosure, including homotrimeric and heterotrimeric polarized trimers, can be fused with other molecules to create multifunctional and / or multispecific proteins. As noted above, various functional moieties, including Fab, Fv scFv, single domain antibodies, peptides,Docket No. BREACH-001 / WOOl cytokines, and / or enzymes can be fused to individual monomers in various configurations to create such proteins.Without wishing to be bound by theory, multispecific or multifunctional molecules disclosed herein can target (e.g., localize, bridge and / or activate) an immune cell (e.g., an immune effector cell chosen from an NK cell, a T cell, a B cell, a dendritic cell or a macrophage), at a cancer cell and / or alter the tumor stroma, e.g., alter the tumor microenvironment near the cancer site. Increasing the proximity and / or activity of the immune cell using the multispecific molecules described herein is expected to enhance an immune response against the cancer cell, thereby providing a more effective cancer therapy. Without being bound by theory, a targeted, localized immune response against the cancer cell is believed to reduce the effects of systemic toxicity of the multispecific molecules described herein. Accordingly, provided herein are multispecific molecules (e.g., multispecific or multifunctional molecules) that include the aforesaid moieties, nucleic acids encoding the same, methods of producing the aforesaid molecules, and methods of treating a cancer using the aforesaid molecules. These multispecific or multifunctional molecules are built upon polarized TNFSF ligand trimers that can be fused to up to six different partners at the first degree of interaction.Accordingly, in one aspect, the disclosure features a multispecific or multifunctional molecule (e.g., polypeptide or nucleic acid encoding the same) built upon a polarized TNFSF ligand trimer. Various TNFSF ligands may be used. In one aspect, the polarized TNFSF ligand trimer corresponds to TNF-alpha (TNFSF2) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to LT-alpha (TNFSF 1) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to LT-beta (TNFSF3) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to OX40L (TNFSF4) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to CD40L (TNFSF5) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to FasL (TNFSF6) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds toDocket No. BREACH-001 / WOOlCD27L (TNFSF7) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to CD30L (TNFSF8) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to 4-1BBL (TNFSF9) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to TRAIL (TNFSF 10) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to RANKL (TNFSF 11) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to TWEAK (TNFSF12) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to APRIL (TNFSF13) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to BAFF (TNFSF13B) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to LIGHT (TNFSF 14) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to TL1A (TNFSF 15) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to GITRL (TNFSF 18) wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to EDA-A1 wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer. In another aspect, the polarized TNFSF ligand trimer corresponds to EDA-A2 wherein each monomer is mutated in such a way that they will interact in a polarized fashion to preferentially associate as a polarized trimer.In one aspect, the multispecific or multifunctional molecule is built upon one of the polarized TNFSF ligand aforementioned and fused with functional moieties taken from the examples below:Docket No. BREACH-001 / WOOl(i) a tumor-targeting moiety, e.g., a first tumor-targeting moiety, that binds to a cancer antigen; and one, two or all of:(ii) an immune cell engager chosen from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager;(iii) a cytokine molecule;(iv) a stromal modifying moiety;(v) a checkpoint inhibitor;(vi) an enzyme; and(vii) a half-life extension moiety.In some embodiments of the aforesaid molecules: if (ii) and (iii) are absent, then (i) and (iv) are present, if one (i) and one (ii) are present, then (iii) or (iv) or both are present, or if one (i) and one (iii) are present, then (ii) or (iv) or both are present.In some embodiments, the multispecific or multifunctional molecule includes (i), (ii) and one or both of (iii) and (iv).In some embodiments, the multispecific or multifunctional molecule includes (i), (iii) and one or both of (ii) and (iv).In some embodiments, the multispecific or multifunctional molecule includes (i), (ii) and (iii). In other embodiments, the multispecific or multifunctional molecule includes (i), (ii) and (iv).In yet another embodiment, the multispecific or multifunctional molecule polypeptide includes (i), (ii), (iii) and (iv).In another aspect, provided herein is a multispecific or multifunctional molecule polypeptide that includes:(i) at least two tumor targeting moieties, e.g., a first and second tumor-targeting moiety, that bind to one or more cancer antigens; and one, two or all of:(ii) an immune cell engager chosen from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager;(iii) a stromal modifying moiety; andDocket No. BREACH-001 / WOOl(iv) a cytokine molecule, e.g., that includes at least two non-contiguous polypeptides (e.g., a multichain cytokine). In some embodiments, the cytokine molecule comprises two chains, e.g., an alpha and beta chain (e.g., IL- 12).In some embodiments, the at least two tumor targeting moieties, e.g., the first and second tumor-targeting moieties, bind to the same or a different cancer antigen.In some embodiments, the multispecific or multifunctional molecule includes one or two immune cell engagers as described herein. In one embodiment, the one or two immune cell engagers binds to and / or inhibits a checkpoint molecule chosen from one or two of CTLA4, PD1, PD-L1, PD- L2, TIM3, LAG3, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), BTLA, KIR, MHC class I, MHC class II, GAL9, VISTA, BTLA, TIGIT, LAIR1, or A2aR. In one embodiment, the multispecific or multifunctional molecule includes two tumor-targeting moieties and one immune cell engager, e.g., a checkpoint binder or CD3. In one embodiment, the multispecific or multifunctional molecule includes two tumor-targeting moieties and two immune cell engagers, e.g., two checkpoint binder (e.g., the same or different checkpoint binder), or a checkpoint binder and CD3.In some embodiments, the first tumor-targeting moiety binds to CD123, the second tumortargeting moiety binds to CD47, and the T cell engager is or comprises a CD3 agonist.In yet another aspect, the multifunctional (e.g., bifunctional) molecule includes a stromal modifying moiety and a tumor-targeting moiety (e.g., an antibody molecule, a ligand molecule, or a receptor molecule) that binds to a tumor antigen or a stromal antigen.In embodiments of any of the aforesaid multispecific or multifunctional molecules, the molecules can further comprise a second tumor-targeting moiety. In embodiments, the second tumortargeting moiety binds to the same or a different cancer antigen as the first tumor-targeting moiety, e.g., the tumor-targeting moiety in (i). The second tumor-targeting moiety binds to a different epitope on the same cancer antigen as the first tumor-targeting moiety. In other embodiments, the second tumor-targeting moiety and the first tumor-targeting moiety bind to different cancer antigens. The different cancer antigens can be present on the same cell or tumor tissue, or can be present on different cells or tumor tissues.In an embodiment a trimeric ligand is multispecific, e.g., it comprises two or more targeting moieties, wherein a first targeting moiety has a binding specificity for a first epitope and a targeting moiety has binding specificity for a second epitope. In an embodiment the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodimentDocket No. BREACH-001 / WOOl the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In an embodiment the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment a multispecific trimeric ligand comprises a third, fourth, fifth, or sixth targeting moiety. In an embodiment, a multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule. As previously described, targeting moieties can comprise a variety of antibody, ligand, or other domains.In an embodiment, a multispecific trimeric ligand according to the disclosure is bispecific and has specificity for no more than two antigens. A bispecific molecule is characterized by a first targeting moiety which has binding specificity for a first epitope and a second targeting moiety that has binding specificity for a second epitope. In an embodiment the first and second epitopes are on the same antigen, e g., the same protein (or subunit of a multimeric protein). In an embodiment the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In an embodiment the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).In an embodiment a bispecific trimeric ligand comprises a first heavy chain variable domain sequence (VH) and a first light chain variable domain sequence (VL) which have binding specificity for a first epitope and a second VH and a second VL which have binding specificity for a second epitope. In an embodiment a bispecific trimeric ligand comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In an embodiment a bispecific trimeric ligand comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In an embodiment a bi specific trimeric ligand comprises a scFv or a Fab, or fragment thereof, have binding specificity for a first epitope and a scFv or a Fab, or fragment thereof, have binding specificity for a second epitope.In yet other embodiments, the immune cell engager and / or the tumor-targeting moiety binds to, but does not inhibit, a checkpoint inhibitor (e.g., a cell, e.g., an immune cell, expressing a checkpoint inhibitor). In other embodiments, the immune cell engager and / or the tumor-targeting moiety binds to, and inhibits, a checkpoint inhibitor (e.g., a cell, e.g., an immune cell, expressing a checkpoint inhibitor). Exemplary checkpoint molecules include, but are not limited to, CTLA4, PD1, PD-L1, PD-L2, TIM3, LAG3, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), BTLA, KIR, MHC class I, MHC class II, GAL9, VISTA, BTLA, TIGIT,Docket No. BREACH-001 / WOOlLAIR1, and A2aR. In one embodiment, the immune cell engager and / or the tumor-targeting moiety binds to, but does not inhibit, a PD1-PDL1 interaction. In another embodiment, the immune cell engager and / or the tumor-targeting moiety binds to and inhibits a PD1-PDL1 interaction.In embodiments of any of the aforesaid multispecific or multifunctional molecules, the molecules can further include a tumor-targeting peptide or tumor-penetrating peptide, such as the iRGD peptide. Tumor-targeting peptides specialized peptides designed to target tumor tissue, e.g., by binding to surface receptors on tumor cells, thus localizing the peptide (and trimeric ligand) to the tumor. Tumor-penetrating peptides go one step further and penetrate tumor tissue, facilitating deeper tissue infiltration through mechanisms such as receptor-mediated endocytosis and protease activation. For example, certain peptides, such as iRGD, initially bind to integrin receptors on tumor blood vessels and subsequently activate a tissue-penetrating pathway that enables them to cross tumor barriers. Thus, incorporating such peptides into a trimeric ligand allows the trimeric ligand to localize with and even penetrate tumors.In any of the embodiments disclosed herein, a multispecific molecule disclosed does not activate an immune cell when a component is presented individually, e g., outside the context of the multispecific molecule (or in the context of a multispecific molecule having an individual component, e.g., an individual immune cell engager); but the multispecific molecule activates the immune cell when presented in the context of a multispecific molecule comprising two or more components, e.g., two or more immune cell engagers. For example, the multispecific molecule can become activated when binding the immune cell when two different receptors are bound by different moieties of the multispecific molecule or when two different epitopes on the same receptor of the effector cells are bound by the multispecific molecule (e.g. activation or inhibition of the corresponding receptor on the immune cell).In embodiments, the multispecific or multifunctional molecule can include two or three tumor targeting moieties to two or three cancer antigens chosen from, e.g., mesothelin, PDL1, HER3, IGF1R, FAP, CD123 or CD47. For example, the first and second tumor targeting moieties are an anti- mesothelin antibody molecule and an anti-PDLl antibody molecule, respectively; or the second and first tumor targeting moieties are an anti-mesothelin antibody molecule and an anti-PDLl antibody molecule, respectively. Other combinations include, but are not limited to, the first and second tumor targeting moieties are an anti -FAP antibody molecule and an anti-PDLl antibody molecule, respectively; or the second and first tumor targeting moieties are an anti-FAP antibody molecule and an anti-PDLl antibody molecule, respectively. In other embodiments, the first and second tumorDocket No. BREACH-001 / WOOl targeting moi eties are an anti-HER3 antibody molecule and an anti -IGF 1R antibody molecule, respectively; or the second and first tumor targeting moieties are an anti-HER3 antibody molecule and an anti-IGFIR antibody molecule, respectively. In other embodiments, the first and second tumor targeting moieties are an anti-CD123 antibody molecule and an anti-CD47 antibody molecule, respectively; or the second and first tumor targeting moieties are an anti-CD123 antibody molecule and an anti-CD47 antibody molecule, respectively.In some embodiments, the multispecific or multifunctional molecule can include an immune cell engager, such as an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager, or a combination thereof. In some embodiments, the immune cell engager comprises an NK cell engager that mediates binding to and activation of, an NK cell. In other embodiments, the immune cell engager comprises an NK cell engager that mediates binding to but not activation of, an NK cell. Exemplary NK cell engagers can be chosen from an antibody molecule, e.g., an antigen binding domain, or ligand that binds to (e.g., activates NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g, CD16a, CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD 100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD 160. In some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen binding domain that binds to NKp30 or NKp46.With reference to FIGS. 1A-B and FIGS. 3A-M, various exemplary multispecific and / or multifunctional trimeric ligands are disclosed. It should be noted that these trimeric ligands are exemplary and in no way limit the scope of the disclosure, which can provide various combinations of functional moieties, as further described herein.Multifunctional and / or multispecific molecules can be created by introducing specific mutations within a TNFSF trimer ligand that results in a polarized trimer. For example, and with reference to FIGS. 1A-B, by introducing paired mutations AEB1, A2:C2, and B3:C3 into individual monomers A, B, and C, the monomers will preferentially associate with one another in a specific configuration through the creation of new covalent and / or non-covalent associations.Functional moieties may also be fused to each of the individual monomers, e.g., up to six functional moieties L1-L6, as shown in FIG. 3A. Each of these fusion partners can be fused to the N- terminus and / or the C-terminus of one or several of the monomers as further described herein. FIG. 1C shows the polarized trimer with the 6 fusion partners, L1-L6. With reference to FIGS. 3A-M, various exemplary multispecific and / or multifunctional trimeric ligands are disclosed. It should beDocket No. BREACH-001 / WOOl noted that these trimeric ligands are exemplary and in no way limit the scope of the disclosure, which can provide various combinations of functional moieties, as further described herein.FIG. 3B illustrates two embodiments of integrating an Fv (fragment, variable region), containing variable domains of both heavy (VH) and light (VL) chains into a trimeric ligand according to the disclosure. In any embodiment, the Fv can represent a binding moiety specific to a tumor antigen, an immune antigen, a stromal antigen, a vascular antigen, and the like. In one embodiment, the VH and VL may be fused to the same monomer (e.g., a VL fused to the N-terminus and a VH fused to the C-terminus), which then associate with one another to form an antigen binding domain (FIG. 2B, top). In another embodiment, the VH and VL are fused to different monomers (e.g., a VL fused to a first monomer and a VH fused to a second monomer), which then associate with one another to form an antigen binding domain. In this embodiment, the association of the VL and VH on separate monomers may also promote the individual monomers within the trimeric ligand to associate in a specific configuration.FIG. 3C illustrates an embodiment of an scFv (single-chain variable fragment) fused to a monomer within a trimeric ligand. In any embodiment, the scFv can represent a binding moiety specific to a tumor antigen, an immune antigen, a stromal antigen, a vascular antigen, and the like. An scFV is a fusion protein that comprises the variable regions of the heavy (VH) and light (VL) chains of an antibody connected by a short peptide linker.FIG. 3D illustrates an embodiment of a VHH (single domain antibody) fused to a monomer within a trimeric ligand. In any embodiment, the VHH can represent a binding moiety specific to a tumor antigen, an immune antigen (e.g., immune cell engager orNK cell engager), a stromal antigen, a vascular antigen, or can act as a half-life extension.FIG. 3E illustrates an embodiment of an Fc (fragment, crystallizable) fused to a trimeric ligand. In this embodiment, the two monomers of the dimeric Fc (i.e., the constant regions of two heavy chains, which pair together to create a single functional form) can be fused to the polarized trimer so that each Fc monomer is fused to an individual TNFSF ligand monomer. In another embodiment, a single-chain Fc dimer can be fused to a single TNFSF ligand monomer. When incorporated into a multifunctional and / or multispecific molecule according to the disclosure, an Fc can provide various beneficial properties such as half-life extension and immune engaging functions, e.g., antibodydependent cellular cytotoxicity (ADCC,) antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).Docket No. BREACH-001 / WOOlFIG. 3F illustrates an embodiment of a peptide fused to a monomer within a trimeric ligand. The peptide can be a tumor-targeting peptide such as the RGD4C, iRGD, LyP-1, K237, IL4RPep-l, mUNO, and the like. Incorporating tumor-targeting peptides into a trimeric ligand according to the disclosure can help localize the trimeric ligand to a tumor.FIG. 3G illustrates an embodiment of an enzyme fused to a monomer within a trimeric ligand. In embodiments, the enzyme can be an extracellular matrix degrading enzyme or a proteolytic enzyme to break the mucosal protein coating that surrounds all malignant tumors to increase the tumor penetration of the molecule, or any enzyme that could have a therapeutic activity.FIG. 3H illustrates an embodiment of a cytokine fused to a monomer within a trimeric ligand. The cytokine can be a pro-inflammatory cytokine, as described herein. In embodiments, the cytokine can be wild-type or muteins of immune activating cytokines, including but not limited to IL-2, IL- 15, IL- 12, IL- 18, IL-21, and the like.FIG. 31 illustrates an embodiment of a VHH and a cleavable mask fused to individual monomers with a trimeric ligand. In such embodiments, the cleavable mask binds to the binding domain of the VHH, but is engineered to have a cleavage site, e.g., specific to matrix metalloproteinase (MMP) proteins. Such proteins are highly expressed in the tumor microenvironment (TME). When the trimeric ligand enters the TME, the mask is cleaved and exposes the binding site of the VHH. Thus, the mask is preferentially cleaved only when in close association with the tumor. Such tumor- activated masks can be used to increase the tumor specificity of a binding domain to minimize any undesired on-target, off-cancer effects of the molecule. In some cases, masking an antibody can also increase the therapeutic window of the molecule.FIG. 3 J illustrates an embodiment of an Fc-antibody fusion molecule fused to a TNFSF ligand monomer. In this embodiment, the Fc-monomer fusion can interact on one hand with complementary polarized TNFSF ligand monomers to form a functional polarized trimer, and on the other hand with a monovalent Fc fusion protein, such as an antibody, a VHH-Fc fusion protein or any other Fc fusion protein. This design leverages the architecture of an Fc fusion protein (such as half-life extension and other Fc-related properties such as immune cell activation described at least with reference to FIG. 3E), and the properties of a TNFSF ligand, as described herein.Various functional moieties can be combined with a trimeric ligand to create new multifunctional and / or multispecific molecules. FIG. 3K illustrates an embodiment of a multispecific and multifunctional molecule that is a T cell engager, checkpoint inhibitor, and cDCl recruiter. As shown in FIG. 3F, this molecule can include six functional moieties, including: (i) a first immune cellDocket No. BREACH-001 / WOOl engager, in this example ant anti-CD3; ; (ii) a first tumor-targeting moiety (TAA1); (iii) a second tumor-targeting moiety (TAA2); (iv) a checkpoint inhibitor ; (v) a cDCl immune cell engager, in this example Clec9A; and (vi) a half-life extension moiety (HLE). Thus, in this embodiment the multifunctional and multispecific molecule can target two different tumor antigens, engage with two different types of immune cells via different mechanisms such as immune cell recruitment, activation, and checkpoint inhibition, and have an extended half-life. In addition, this multifunctional T cell engager is built upon a TNFSF ligand such as LIGHT that possesses immune cell modulation properties such as T cell co-stimulation. Such a type of multifunctional design can address multiple interconnected biological mechanisms and provide an integrated response to diseases such as cancer.FIG. 3L illustrates an embodiment of a multispecific and multifunctional molecule that is a NK cell engager and activator and checkpoint inhibitor. As shown in FIG. 3L, this molecule can include six functional moieties, including: (i) a first immune cell engager, in this example a NK cell engager (CD16) ; (ii) a first tumor-targeting moiety (TAA1); (iii) a second tumor-targeting moiety (TAA2); (iv) a checkpoint inhibitor; (v) a NK cell-stimulating cytokine, in this example IL-15; and (vi) a half-life extension moiety (HLE). Thus, in this embodiment the multifunctional and multispecific molecule can target two different tumor antigens, engage with two different types of immune cells, interact with cytokine receptors, and also have an extended half-life. In addition, this multifunctional NK cell engager is built upon a TNFSF ligand (such as LIGHT) whose properties can complement the multifunctional protein, including but not limited to: Immune stimulation, such as T cell and NK cell activation through HVEM, that can enhance cytotoxicity; promoting DC maturation and function; and increasing infiltration of immune cells into the tumor. LIGHT also has beneficial vascular effects, including normalizing of tumor vasculature, with a potential to improve drug delivery to the tumor. Additionally, LIGHT can perform stromal remodeling. LIGHT can reduce tumor- associated fibrosis and could lead to improved tumor access, thereby enhancing therapeutic protein penetration. Together, these mechanisms can address both immunosuppression and vascular barriers, while also improving response durability.FIG. 3M illustrates an embodiment of a multispecific and multifunctional molecule that is a tumor targeted and activated immune activator. As shown in FIG. 2M, this molecule can include six functional moieties, including: (i) stromal antigen binder to promote cancer targeting, ; (ii), an immune modulator such as a checkpoint inhibitor (CPI); (iii) a half-life extension moiety (HLE); (iv) an immune cell activating cytokine, such as IL-2, IL-12, or IL-15; (v) a cleavable mask which masks the functional component of the cytokine outside of the tumor microenvironement; and (vi) a tumor-Docket No. BREACH-001 / WOOl targeting peptide. Thus, in this embodiment the multifunctional and multispecific molecule can target a tumor, activate its cytokine only within the tumor environment, and engage with immune cells, thereby targeting a tumor and activating patient immune response. In addition, this multifunctional molecule is built upon a TNFSF ligand (such as LIGHT) whose properties can complement the multifunctional protein, including but not limited to: Immune stimulation, such as T cell and NK cell activation through HVEM, that can enhance cytotoxicity; promoting DC maturation and function; and increasing infiltration of immune cells into the tumor. LIGHT also has beneficial vascular effects, including normalizing of tumor vasculature, with a potential to improve drug delivery to the tumor. Additionally, LIGHT can perform stromal remodeling. LIGHT can reduce tumor-associated fibrosis and could lead to improved tumor access, thereby enhancing therapeutic protein penetration. Together, these mechanisms can address both immunosuppression and vascular barriers, while also improving response durability.The ability to incorporate up to six first-degree fusions in trimeric ligands as described herein enables multiple new modalities. Individual monomers with fused functional moieties may be considered as “modules” that can be mixed and matched together to form new multifunctional and / or multispecific proteins. FIGS. 3N-P illustrate several examples of how IgG-based formats (such as antibodies, multi-specific antibodies, and immune cell engagers) can be reformatted using polarized trimeric ligands and functional moieties as described herein. For example, as shown in FIG. 3N, the VH and VL domains of an antibody can be fused to a monomer of a TNFSF ligand trimer. Such a trimer may also incorporate a half-life extension moiety and a tumor targeting peptide to improve its therapeutic potential. As shown in FIG. 30, a multi-specific antibody can be reformatted by incorporating a binder to a second antigen which is fused to another monomer in the trimeric ligand. While in this embodiment, the binder to the second antigen replaces the half-life extension moiety, the half-life extension moiety may also be included as the trimeric ligand may include up to six functional units. As shown in FIG. 3P, an immune cell engager (e.g., a T cell or NK cell engager) can be reformatted with a trimeric ligand by fusing binders to a tumor antigen (TAA) to one or two or more monomers, and a binder to an immune cell (such as anti-CD3 or anti-CD16) fused to another monomer to replicate the same function, with the potential for an additional functional unit to be incorporated.FIGS. 3A-P illustrate various embodiments of multispecific and multifunctional molecules, comprising TNFSF ligand trimers with various functional moieties attached thereto. While these embodiments depict specific examples of multispecific and multifunctional molecules, it should beDocket No. BREACH-001 / WOOl noted that the present disclosure is not so limited, and can encompass various combinations of different TNFSF ligand trimers with new covalent and / or non-covalent associations and functional units attached thereto.(g) Further aspectsIn another aspect, the disclosure provides a vector, e.g., an expression vector, comprising one or more of any nucleic acid molecules described herein.In another aspect, the disclosure provides a host cell comprising a nucleic acid molecule or a vector described herein.In another aspect, the disclosure provides a method of making, e.g., producing, a multispecific or multifunctional molecule polypeptide described herein, comprising culturing a host cell described herein, under suitable conditions, e.g., conditions suitable for gene expression and / or homo- or heterodimerization.In another aspect, the disclosure provides a pharmaceutical composition comprising a multispecific or multifunctional molecule polypeptide described herein and a pharmaceutically acceptable carrier, excipient, or stabilizer.In another aspect, the disclosure provides a method of treating a cancer, comprising administering to a subject in need thereof a multispecific or multifunctional molecule polypeptide described herein, wherein the multispecific antibody is administered in an amount effective to treat the cancer.In some embodiments, the cancer is a solid tumor cancer, or a metastatic lesion. In some embodiments, the solid tumor cancer is one or more of pancreatic (e.g., pancreatic adenocarcinoma), breast, colorectal, lung (e g., small or non-small cell lung cancer), skin, ovarian, or liver cancer. In some embodiments, the cancer is a hematological cancer.In some embodiments, the method further comprises administering a second therapeutic treatment. In some embodiments, second therapeutic treatment comprises a therapeutic agent (e.g., a chemotherapeutic agent, a biologic agent, hormonal therapy), radiation, or surgery. In some embodiments, therapeutic agent is selected from: a chemotherapeutic agent, or a biologic agent.IV. Nucleic Acid Molecules, Vectors, Host Cells, and Pharmaceutical CompositionsProvided herein are nucleic acid molecules encoding polarized TNFSF ligand timers, and related fusion proteins. In some embodiments, the nucleic acid molecules are provided as a DNA sequence. In other embodiments, nucleic acid molecules are provided as a messenger RNA transcript.Docket No. BREACH-001 / WOOlNucleic acid molecules can be prepared using known methods. In certain embodiments, a variant polarized TNFSF ligand trimer, or a fusion protein can be encoded by a nucleic acid sequence. Such a nucleic acid sequence can be included within an appropriate vector for expression and secretion using recombinant and / or synthetic techniques. With regard to a multispecific and multifunctional protein described herein, it will be understood that each of the first and the second, third, fourth, fifth, sixth (and, optionally additional) domain can be encoded by nucleic acid sequences on the same vector or different vectors. If a linker is to be present in a multispecific protein, it can be encoded by a nucleic acid sequence and be operably linked to the first domain or the second domain (and optionally, a third and / or fourth domain). The nucleic acid sequences can be operably linked to one or more of the following: a signal peptide, a promoter, a transcription terminator, a polyA sequence, etc. for appropriate expression in a host cell. Other domains (e.g., half-life extension domains), can also be incorporated into one or more nucleic acid sequence(s) for expression. Depending on the vector system and host cell utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. The promoter can be selected such that it drives the expression of the polynucleotide in the respective host cell.A nucleic acid molecule encoding a multifunctional and / or multi specific protein of the present disclosure can be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed, e.g., by Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel, 1987, 1993, and can be used to construct nucleic acid sequences which encode a binding protein. Accordingly, the disclosure provides for a vector comprising the isolated nucleic acids set forth herein. Vectors of particular interest include plasmids, phagemids, phage derivatives, viruses (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like), and cosmids.Once isolated, a polynucleotide sequence may be placed into an expression vector, which is then transfected into a host cell such as, for example, an E. coli cell, a simian COS cell, a human embryonic kidney 293 cell (e.g., 293E cell), a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce a binding protein, to obtain the synthesis of binding proteins in the recombinant host cells. Recombinant production of binding proteins is well known in the art. Expression control sequences (e.g., regulatory sequences) refer to DNA sequences necessary for theDocket No. BREACH-001 / WOOl expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to optionally utilize promoters, polyadenylation signals, and / or enhancers, if needed.A variety of expression vector / host systems may be utilized to contain and express the polynucleotide encoding the variant polarized TNFSF ligand trimer, multispecific protein, or fusion protein. Examples of expression vectors for expression in E.coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1): 111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells, PICHIAPINK™ Yeast Expression Systems (Invitrogen), BACUVANCE™ Baculovirus Expression System (GenScript), etc.Thus, the variant polarized TNFSF ligand trimer, multispecific proteins, or fusion proteins as described herein, in some embodiments, are produced by introducing a vector encoding the protein as described above into a host cell and culturing said host cell under conditions whereby the protein domains are expressed, and which may be isolated and, optionally, further purified.Any of the variant variant polarized TNFSF ligand trimer, multispecific binding protein, or fusion protein, multispecific binding proteins, fusion proteins, or nucleic acid molecules encoding such proteins described herein can be non-naturally occurring (e.g., synthetic, recombinant, isolated, substantially purified, etc.). In one instance, the binding protein or nucleic acid molecule is synthetic. In another instance, the binding protein or nucleic acid molecule is recombinant. In another instance, the binding protein or nucleic acid molecule is isolated. In another instance, the binding protein or nucleic acid molecule is substantially purified.V. Treatments and Methods of UseIn one aspect, provided herein are methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a multifunctional and / or multispecific polarized TNFSF ligand trimer fusion protein. Once made, the subject variant variant polarized TNFSF ligand trimer, multispecific binding protein, or fusion protein, multispecific binding proteins, and fusion proteins find use in a number of oncology applications, such as by promoting immune cell activation and engagement (cytotoxicity) of cancer cells. Accordingly, the subject variant polarized TNFSF trimer, multispecific binding protein, or fusion protein, multispecific binding proteins, and Fc fusion proteins find use in the treatment of these cancers. a. Formulations for In Vivo AdministrationDocket No. BREACH-001 / WOOlProvided herein is a pharmaceutical composition comprising (a) a variant variant polarized TNFSF ligand trimer, multispecific binding protein, or fusion protein, a multispecific binding protein, a nucleic acid molecule encoding the protein, a vector, or a host cell described herein, and (b) a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia (U.S.P.) or other generally recognized pharmacopeia for use in animals, including humans. A “pharmaceutically acceptable carrier” refers to a carrier that can be administered to a subject, together with an active agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.Formulations of the variant polarized TNFSF ligand trimer, multispecific binding protein, or fusion protein, multi-specific binding proteins, used in accordance with the present disclosure may be prepared for storage by mixing a protein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (as generally outlined in Remington’s Pharmaceutical Sciences 16thedition, Osol, A. Ed.

[1980] ), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, buffers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben, catechol, resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, di saccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). b. Therapeutic UsesThe variant polarized TNFSF ligand trimer, multispecific binding protein, or fusion protein, multispecific binding proteins, and fusion proteins described herein find use in treating patients, such as human subjects, generally with a condition associated with impaired immune response. The term “treatment” as used herein, refers to both therapeutic treatment and prophylactic or preventativeDocket No. BREACH-001 / WOOl measures, which in this example relates to treatment of cancer. Those in need of treatment include those already with cancer as well as those in which the cancer is to be prevented. Hence, the mammal to be treated herein may have been diagnosed as having cancer or may be predisposed or susceptible to the cancer. As used herein the term “treating” refers to preventing, delaying the onset of, curing, reversing, attenuating, alleviating, minimizing, suppressing, halting the deleterious effects or stabilizing of discernible symptoms of the above-described cancerous diseases, disorders or conditions. It also includes managing cancer as described above. By “manage” it is meant reducing the severity of the disease, reducing the frequency of episodes of the disease, reducing the duration of such episodes, reducing the severity of such episodes, slowing / reducing cancer cell growth or proliferation, slowing progression of at least one symptom, amelioration of at least one measurable physical parameter and the like. For example, immunostimulatory variant polarized TNFSF ligand trimer, multispecific binding protein, or fusion protein should promote T cells, NK cells, NKT cells, Myeloid cells, Dendritic cells, MAIT T cells, 78 T cells, and / or innate lymphoid cells (ILCs), or cytokine immunity against target cells, e.g., cancer, infected or pathogen cells and thereby treat cancer or infectious diseases by depleting the cells involved in the disease condition.The variant polarized TNFSF ligand trimer, multispecific binding protein, or fusion protein, multispecific binding proteins, and fusion proteins as disclosed herein are provided in therapeutically effective dosages. A “therapeutically effective dosage” of a variant polarized TNFSF ligand trimer, multi specific binding protein, or fusion protein according to at least some embodiments of the present disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in lifespan, disease remission, or a prevention or reduction of impairment or disability due to the disease affliction. For example, for the treatment of tumors, a “therapeutically effective dosage” preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such as inhibition in vitro by assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject.Docket No. BREACH-001 / WOOlOne of ordinary skill in the art would be able to determine a therapeutically effective amount based on such factors as the subject’s size, the severity of the subject’s symptoms, and the particular composition or route of administration selected. i. Cancer TreatmentThe polarized TNFSF ligand trimers, multispecific and / or multifunctional proteins, or fusion proteins as disclosed herein, alone or in combination with other therapeutic agents, find particular use in the treatment of cancer. In general, embodiments of the disclosure are immunomodulatory, in that rather than directly attack cancerous cells, polarized TNFSF ligand trimer, multispecific proteins, or fusion proteins as described herein stimulate the immune system, and bridge the immune cells to the cancer cells. Thus, unlike tumor-targeted therapies, which are aimed at inhibiting molecular pathways that are crucial for tumor growth and development, and / or depleting tumor cells, cancer immunotherapy is aimed to stimulate the patient’s own immune system to eliminate cancer cells, providing long-lived tumor destruction. Various approaches can be used in cancer immunotherapy, among them are therapeutic cancer vaccines to induce tumor-specific T cell responses, and immunostimulatory antibodies (i.e., antagonists of inhibitory receptors = immune checkpoints) to remove immunosuppressive pathways.Clinical responses with targeted therapy or conventional anti-cancer therapies tend to be transient as cancer cells develop resistance, and tumor recurrence takes place. However, the clinical use of cancer immunotherapy in the past few years has shown that this type of therapy can have durable clinical responses, showing dramatic impact on long term survival. However, although responses are long term, only a small number of patients respond (as opposed to conventional or targeted therapy, where a large number of patients respond, but responses are transient).By the time a tumor is detected clinically, it has already evaded the immune-defense system by acquiring immunoresistant and immunosuppressive properties and creating an immunosuppressive tumor microenvironment through various mechanisms and a variety of immune cells.Accordingly, polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein are useful in treating cancer. Due to the nature of an immune-oncology mechanism of action, polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein de-suppress / activate T cells, NK cells, NKT cells, Myeloid cells, Dendritic cells, MAIT T cells, 78 T cells, and / or innate lymphoid cells (ILCs) activation, and bridge immune cells to cancer cells, such that the immune system will go after the cancer.Docket No. BREACH-001 / WOOl“Cancer,” as used herein, refers broadly to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division causing malignant growth or tumor (e.g., unregulated cell growth). The term “cancer” or “cancerous” as used herein should be understood to encompass any neoplastic disease (whether invasive, noninvasive or metastatic) which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor, nonlimiting examples of which are described herein. This includes any physiological condition in mammals that is typically characterized by unregulated cell growth.“Cancer therapy” herein refers to any method that prevents or treats cancer or ameliorates one or more of the symptoms of cancer. Typically, such therapies will comprise administration of immunostimulatory polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein either alone or in combination with chemotherapy or radiotherapy or other biologies and for enhancing the activity thereof, i.e., in individuals wherein91mmune91tered91n of the immune system suppresses antitumor responses and the efficacy of chemotherapy or radiotherapy or biologic efficacy.The polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein, may be used as a monotherapy or as part of a combination therapy as described herein, can be used in the treatment of solid tumors (including, for example, cancers of the lung, liver, breast, brain, GI tract) and blood cancers (including for example, leukemia and preleukemic disorders, lymphoma, plasma cell disorders) carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. In some embodiments, the cancer is early. In some embodiments, the cancer is advanced (including metastatic). In some embodiments, the cancer for treatment using polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins of the present disclosure includes vascularized tumors, melanoma, nonmelanoma skin cancer (squamous and basal cell carcinoma), mesothelioma, squamous cell cancer, lung cancer, small-cell lung cancer, non-small cell lung cancer, neuroendocrine lung cancer (including pleural mesothelioma, neuroendocrine lung carcinoma), NSCL (large cell), NSCLC large cell adenocarcinoma, small cell lung carcinoma (SCLC), non-small cell lung carcinoma (NSCLC), NSCLC squamous cell, soft-tissue sarcoma, Kaposi’s sarcoma, adenocarcinoma of the lung, squamous carcinoma of the lung, NSCLC with PDLI >=50% TPS, neuroendocrine lung carcinoma, atypical carcinoid lung cancer, cancer of the peritoneum, esophageal cancer, hepatocellular cancer, liver cancer (including HCC), gastric cancer, stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, urothelial cancer, bladder cancer, hepatoma, glioma, brain cancer (as well as edema, such asDocket No. BREACH-001 / WOOl that associated with brain tumors), breast cancer (including, for example, triple negative breast cancer), testis cancer, testicular germ cell tumors, colon cancer, colorectal cancer (CRC), colorectal cancer MSS (MSS-CRC); refractory MSS colorectal; MSS (microsatellite stable status), primary peritoneal cancer, primary peritoneal ovarian carcinoma, microsatellite stable primary peritoneal cancer, platinum resistant microsatellite stable primary peritoneal cancer, CRC (MSS unknown), rectal cancer, endometrial cancer (including endometrial carcinoma), uterine carcinoma, salivary gland carcinoma, kidney cancer, renal cell cancer (RCC), renal cell carcinoma (RCC), gastroesophageal junction cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, carcinoid carcinoma, head and neck cancer, B-cell lymphoma (including non-Hodgkin’s lymphoma, as well as low grade / follicular non-Hodgkin’s lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade / follicular NHL, intermediate grade diffuse NHL, Diffuse Large B cell lymphoma, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom’s Macroglobulinemia, Hodgkin’s lymphoma (HD), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), T cell Acute Lymphoblastic Leukemia (T-ALL), Acute myeloid leukemia (AML), Hairy cell leukemia, chronic myeloblastic leukemia, multiple myeloma, posttransplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, Meigs’ syndrome, Merkel Cell cancer, MSLhigh cancer, KRAS mutant tumors, adult T-cell leukemia / lymphoma, adenoid cystic cancer (including adenoid cystic carcinoma), melanoma, malignant melanoma, metastatic melanoma, pancreatic cancer, pancreatic adenocarcinoma, ovarian cancer (including ovarian carcinoma), pleural mesothelioma, cervical squamous cell carcinoma (cervical SCC), anal squamous cell carcinoma (anal SCC), carcinoma of unknown primary, gallbladder cancer, pleural mesothelioma, chordoma, endometrial sarcoma, chondrosarcoma, uterine sarcoma, uveal melanoma, amyloidosis, AL-amyloidosis, astrocytoma, and / or Myelodysplastic syndromes (MDS).In some embodiments, the cancer for treatment using the polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein includes cancer selected from the group consisting of renal clear cell carcinoma (RCC), lung cancer, NSCLC, lung adenocarcinoma, lung squamous cell carcinoma, gastric adenocarcinoma, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer (TNBC), head and neck tumor, colorectal adenocarcinoma, melanoma, and metastatic melanoma. ii. Variant polarized TNFSF ligand trimer fusion protein MonotherapiesDocket No. BREACH-001 / WOOlThe polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein find particular use in the treatment of cancer as a monotherapy. Due to the nature of immuno-oncology mechanism of action, the goal is to have the the polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein de-suppress / activate T cell and NK cell activation, and bridge the immune cells to cancer cells, such that the immune system will go after the cancers. Any of the the polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein finds use as a monotherapy. iii. Variant polarized TNFSF ligand trimer fusion protein Combination TherapiesAs is known in the art, combination therapies comprising a therapeutic agent targeting an immunotherapy target and an additional therapeutic agent, specific for the disease condition, are showing great promise. For example, in the field of immunotherapy, there are a number of promising combination therapies using a chemotherapeutic agent (either a small molecule drug or an anti-tumor antibody) or with an93mmuneo-oncology antibody.The terms “in combination with” and “co-administration” are not limited to the administration of said prophylactic or therapeutic agents at exactly the same time. Instead, it is meant that the protein or antibody and the other agent or agents are administered in a sequence and within a time interval such that they may act together to provide a benefit that is increased versus treatment with only either the antibody of the present disclosure or the other agent or agents. It is preferred that the antibody and the other agent or agents act additively, and especially preferred that they act synergistically.Accordingly, the polarized TNFSF ligand trimer, multispecific proteins, or fusion proteins as described herein may be administered concomitantly with one or more other therapeutic regimens or agents. In some embodiments, the antibodies of the present disclosure are administered in the same formulation with one or more other therapeutic regimens or agents. In some embodiments, the antibodies of the present disclosure are administered in a separate and / or different formulation from the one or more other therapeutic regimens or agents. The additional therapeutic regimes or agents may be used to improve the efficacy or safety of the antibody. Also, the additional therapeutic regimes or agents may be used to treat the same disease or a co-morbidity rather than to alter the action of the antibody. For example, an antibody or protein as disclosed herein may be administered to the patient along with chemotherapy, radiation therapy, or both chemotherapy and radiation therapy.In some embodiments, the polarized TNFSF ligand trimer, multispecific proteins, or fusion proteins as described herein can be combined with one of a number of checkpoint receptor antibodies. In some embodiments, a patient’s tumor may be evaluated for expression of receptors and the resultsDocket No. BREACH-001 / WOOl then used to inform a clinician as to which antibodies to administer. Any of the the polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein finds use as part of a combination therapy.In some embodiments, the combination or composition further comprises an additional active agent, e.g., a second antigen binding protein. Optionally, the second antigen binding protein binds to a negative regulator of the immune system, an immune suppressor, or an immune checkpoint protein, including but not limited to PD-I, PD-L I, CTLA-4, PDL2, B7-H3, B7-H4, CEACAM-I, TIGIT, PVR, LAG3, CD112, PVRIG, CD96, TIM3, and / or BTLA, or co-stimulatory receptor: ICOS, 0X40, 41BB, CD27, and / or GITR.In some embodiments, the polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein are used in combination with an antibody to an immune checkpoint inhibitor protein. In some embodiments, the immune checkpoint inhibitor protein is selected from the group consisting of an anti-PVRIG antibody, an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti- TIGIT antibody, an anti-CTLA-4 antibody, an anti-PD-L2 antibody, an anti-B7-H3 antibody, an anti B7-H4 antibody, an anti-CEACAM-1 antibody, an anti-PVR antibody, an anti-LAG3 antibody, an anti-CD112 antibody, an anti-CD96 antibody, an anti-TIM3 antibody, an anti-BTLA antibody, an anti- ICOS antibody, an anti-OX40 antibody, or an anti -4 IBB antibody, an anti-CD27 antibody, or an anti-GITR antibody.The polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein may be administered in combination with one or more other prophylactic or therapeutic agents, including but not limited to cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti -angiogenic agents, cardioprotectants, immunostimulatory agents, immunosuppressive agents, agents that promote proliferation of hematological cells, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, or other therapeutic agents.In some embodiments, the chemotherapeutic agent is selected from the group consisting of Platinum, Oxaliplatin, Cisplatin, Paclitaxel (taxol), Sorafenib, Doxorubicin, Sorafenib, 5-FU, and Gemcitabine, Irinotecan (CPT-11).In some embodiments, the other therapeutic is an agent used in radiation therapy for the treatment of cancer. Accordingly, in some embodiments, the active agents described herein are administered in combination with one or more of platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids and difluoronucleosides.Docket No. BREACH-001 / WOOlIn some embodiments, the polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein are used in combination with one or more inflammasome activators. In some embodiments, the inflammasome activator is a CD39 inhibitor. In some embodiments, the CD39 inhibitor is an anti-CD39 antibody.According to at least some embodiments, the the polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein could be used in combination with any of the known in the art standard of care cancer treatment (as can be found, for example, on the World Wide Web at cancer.gov / cancertopics).Administration of the pharmaceutical composition comprising the variant the polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as described herein, preferably in the form of a sterile aqueous solution, may be done in a variety of ways. As is known in the art, protein therapeutics are often delivered by IV infusion. The antibodies of the present disclosure may also be delivered using such methods. For example, administration may be veinous or by intravenous infusion with 0.9% sodium chloride as an infusion vehicle. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 16thedition, Osol, A. Ed., 1980.The dosing amounts and frequencies of administration are, in some embodiments, selected to be therapeutically or prophylactically effective. As is known in the art, adjustments for protein degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. In order to treat a patient, a therapeutically effective dose of the the polarized TNFSF ligand trimer, multispecific binding proteins, or fusion proteins as disclosed herein may be administered. By “therapeutically effective dose” herein is meant a dose that produces the effects for which it is administered.EXAMPLESThe application may be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the application. The following examples are presented in order to more fully illustrate embodiments and should in no way be construed, however, as limiting the broad scope of the application.Example 1 : General strategy for the design of polarized TNFSF ligand trimersThe inventor carried out structure-based identification of residues that are in close proximity between monomers in a trimeric ligand using publicly available crystal structures in the Protein DataDocket No. BREACH-001 / WOOlBank (PDB), and that are not predicted to participate in the binding of their cognate receptors. As shown in FIG. IB, the general strategy to obtain polarized trimers is to create paired mutations in individual monomers (monomers are labeled A, B, and C). For example, as shown in FIG. IB, possible paired mutations include A1 :B1, A2:C2, and B3:C3. The positions for these mutations are selected based on proximity to one another within the trimeric ligand. These paired mutations can introduce new covalent and / or non-covalent associations between the monomers, e.g., by the introduction of new cysteine residues to create disulfide bonds, or other new residues to create salt bridges or hydrophobic interactions. This strategy forces polarization, i.e., monomers will preferentially interact with specific subunits that have reciprocal / complementary mutations. By introducing complementary mutations into the monomers, monomer A interacts with B and C but not with a separate A molecule, or with a BB dimer, or with a CC dimer. Specifically, mutation Al will interact with Bl and A2 will interact with C2. Monomer B will interact with A and C via mutation Bl interacting with Al and mutation B3 interacting with C3. Monomer C will interact with A and B via mutation C2 interacting with A2 and C3 interacting with B3. While this example describes a trimeric ligand with three sets of paired mutations, only a single pair (e.g., Al :B1, A2:C2, and B3:C3) is sufficient to create a polarized trimeric ligand.Potential positions to introduce paired mutations were identified using a computational approach. Specifically, identified residues were mutated in silico and new interactions between the monomers were tested in silico using AlphaFol d2 (DeepMind). Mutated sequences that exhibited satisfactory folding and monomer-monomer interactions in silico were selected for expression in mammalian cells. Polarized trimerization was confirmed by behavior of the protein as examined by SDS-PAGE and SEC-HPLC.Example 2: Characterization of Wild-type LIGHTWild-type LIGHT (TNFSF14) protein was expressed and purified from CHO cells. To investigate the structural properties of LIGHT, the protein was subjected to SDS-PAGE analysis under both reducing and non-reducing conditions.Purified wild-type LIGHT was divided into two aliquots. The first aliquot was mixed with SDS-PAGE sample buffer containing dithiothreitol (DTT) as a reducing agent to break any disulfide bonds between subunits. The second aliquot was mixed with SDS-PAGE sample buffer without reducing agents, maintaining the protein’s disulfide bond integrity. Both samples were heated at 95C for 5 minutes prior to electrophoresis.Docket No. BREACH-001 / WOOlThe samples were then loaded into a 12% SDS-PAGE gel and run under standard conditions alongside a molecular weight ladder. Following electrophoresis, the gel was stained with Coomassie Brilliant Blue to visualize protein bands.In both reducing and non-reducing conditions, a single band corresponding to a molecular weight of approximately 25 kDa was observed (FIG. 4A). No higher molecular weight bands, which would indicate the presence of oligomeric forms such as dimers or trimers, were observed under nonreducing conditions. These results show that the wild-type LIGHT protein, although known to form trimers in its native state, readily disassociates into monomers under denaturing conditions, regardless of the presence or absence of reducing agents. This suggests that the trimer form of LIGHT is not stabilized by disulfide bonds and can easily dissociate during SDS-PAGE.The absence of disulfide-linked oligomers indicates that LIGHT’s trimerization is mediated by non-covalent interactions rather than disulfide bonds, allowing the trimer to disassemble into monomers under denaturing conditions.Table 11: Properties of Wild-Type LIGHTExample 3: SDS-PAGE and Size-Exclusion Chromatography (SEC) Analysis of Mutant and Wild- Type LIGHT Interactions (Single Covalent Association between Two Monomers)Two distinct LIGHT (TNFSF 14) muteins were generated to assess their ability to interact with wild-type LIGHT and form stable oligomers by including a single pair of complementary mutations between the two muteins. The first trimeric ligand (BB112) included two monomers (A, B) having mutations A:S182C and B:S200C, and the second trimeric ligand (BB113) two monomers (B, C) having mutations B:G188C and C:T191C. These paired mutations were selected at positions in the binding interface between LIGHT monomers which are likely to form a disulfide bond due to their close proximity, thus increasing stability of the oligomer and promoting a particular configuration of the monomers within the trimeric ligand. Specifically, the monomers A and B will form a dimer through a new disulfide bond, which will then form a trimer together with the WT monomer. Thus, the resulting oligomer may be polarized, in that the three LIGHT molecules (mutein A, mutein B, and WT) will tend to associate in the same fashion.Docket No. BREACH-001 / WOOlA third mutein (BB127, see FIG. 4B) was also generated with the same paired mutations as BB113 (B: G188C and C:T191C), and including additional single mutations at F202 in all three monomers (A:F202Y; B: G188C / F202Y; C:T191C / F202Y). Without being bound by theory, the mutation of Phe202 to Tyr202 (F202Y) can create additional interchain hydrogen bounds, thus further stabilizing the trimeric ligand.The proteins were expressed and purified from CHO cells and subjected to SDS-PAGE analysis as described in the previous example. As shown in FIG. 4B, in both reducing and nonreducing conditions, a band corresponding to a molecular weight of approximately 25 kDa (monomer) was observed. Additionally, in non-reducing conditions, a higher molecular weight band was observed at approximately 40 kDa, corresponding to a dimeric form of the two mutein LIGHT molecules linked by a disulfide bond introduced from the single pair of mutations within two of the monomers.Protein purity was also assessed by SEC-HPLC. The profiles of the mutant and wild-type LIGHT in solution shows a prominent peak around 9 minutes in line with the expected molecular weight of a LIGHT trimer. Taken together, the SDS-PAGE and SEC-HPLC results confirm the presence of covalent dimers in line with the formation of the new inter-chain disulfide bounds, and indicate that the muteins are found predominantly as polarized trimers in solution.As shown in Table 12, below, most of the expressed muteins had good purity, stability, and yield, representing good candidates for multifunctional and / or multispecific trimeric ligands. Additionally, in the third mutein (BB127), the addition of F202Y mutations to all monomers, through the introduction of additional interchain hydrogen bounds, led to increased purity, stability and increased expression yield. Thus, a single new covalent interaction (G188C / T191C) may be sufficient for efficient polarization.Table 12: Properties of LIGHT Muteins with a Single New Covalent Association Between Two MonomersDocket No. BREACH-001 / WOOlExample 4: SDS-PAGE and SEC Analysis of Mutant and Wild-Type LIGHT Interactions (Two Covalent and / or Non-Covalent Associations linking One Monomer to Two Monomers)LIGHT (TNFSF14) muteins were generated to assess their ability to interact with one another to form stable and polarized trimers. In this example, two new covalent and / or non-covalent associations were introduced between one monomer and the other two monomers, i.e., A:B and B:C. Thus, each of the monomers contains at least one mutation relative to wild-type.Three trimeric ligands were designed. The first trimeric ligand (BB 114) with monomers A, B, C included mutations A: S182C, B: S200C / G188C, and C: T191C, which are intended to form disulfide bonds between monomers A and B via paired mutations S182C / S200C and between monomers B and C via paired mutations G188C / T191C. The second trimeric ligand (BB173) included additional F202Y mutations in all monomers (A: S182C / F202Y; B: S200C / G188C / F202Y; C: T191C / F202Y). The third trimeric ligand (BB 174) included mutations at the same positions as BB 173, but replaced the S182C / S200C paired mutations with Lysine and Aspartic acid residues: A: S182K / F202Y; B: G188C / S200D / F202Y; C: T191C / F202Y (BB174). These are intended to create a non-covalent association (here, a salt bridge) between monomers A and B instead of a covalent association (disulfide bond), while also including a disulfide bond between monomers B and C.The mutations were selected at positions in the association interface between LIGHT monomers and thus are likely to form disulfide bonds and salt bridges and increase stability of the oligomer. Mutations of Phe202 into a Tyr, through the introduction of additional interchain hydrogen bounds led to increased purity, stability and increase expression yield.The proteins were expressed, purified, and analyzed using SDS-PAGE as previously described. A band of 25 kDa (corresponding to single monomers disassociated from one another) was observed in reducing conditions. In non-reducing conditions, three bands were observed: at 25 kDa (monomer), 40 kDa (dimer), and 60 kDa (trimer). As shown in the chromatographs, the trimeric form of the oligomer is exceedingly stable and represents the majority of the sample. Thus, the addition of the third mutant LIGHT molecule resulted in disulfide bonds covalently attaching each of the mutant monomers to one another, resulting in a highly stable and polarized trimer.Purity, stability, and expression yield of these three muteins is in Table 13, below.Docket No. BREACH-001 / WOOlTable 13: Properties of LIGHT Muteins with Covalent and / or Non-Covalent Associations Between One Monomer and Two MonomersExample 5: SDS-PAGE and SEC Analysis of Mutant and Wild-Type LIGHT Interactions (Three Covalent and / or Non-Covalent Associations to Create Fully Connected Monomers)Several distinct LIGHT (TNFSF14) muteins were generated to assess their ability to form a polarized trimer and interact in a stable fashion. In this example, three new covalent and / or non- covalent associations were introduced between each of the monomers to create a fully connected trimeric ligand, i.e., A:B, B:A, A:C. Like in the previous example, each of the monomers in each trimeric ligand contain at least one mutation relative to wild-type.The mutations were selected at positions in the association interface between LIGHT monomers and thus are likely to form disulfide bonds and salt bridges and increase stability of the oligomer. Among these, one mutein (BB 121) included monomers having mutations A: S182C / S200D; B: S182C / S200C; C: S182K / S200C. Another mutein (BB114) included monomers having mutations A: S182C; B: S200C / G188C / R124A; C: T191C / V240D. Another mutein (BB 144) included mutations A: S182C / S200D; B: S182C / S200C; C: S182R / S200C. Each of the muteins were designed to include two disulfide bonds and one salt bridge to fully connect each of the monomers, i.e., each of the monomers includes two mutations that are paired to a mutation in one of the other monomers.In the case of the V240D mutation (BB114), the mutation to an aspartate residue at position 240 is designed to interact with the naturally occurring arginine residue at position 124. To avoid nondesired interactions and promote proper polarization, R124 is mutated into an alanine in the relevant monomer.The proteins were expressed, purified, and analyzed using SDS-PAGE as previously described. For BB121 and BB141, a band of 25 kDa (corresponding to single monomers disassociated from one another) was observed in reducing conditions. In non-reducing conditions, three bands were observed: at 25 kDa (monomer), 40 kDa (dimer), and 60 kDa (trimer). As shown in the chromatographs, the trimeric form of the oligomer is exceedingly stable and represents the majority of the sample. In contrast, BB115 included multiple bands and a diverse chromatograph, indicatingDocket No. BREACH-001 / WOOl multiple off-target effects resulting in undesired oligomers. Thus, creating a mutant LIGHT molecule with fully connected monomers can result in a highly stable and polarized trimer.Purity, stability, and expression yield of these three muteins is in Table 14, below.Table 14: Properties of LIGHT Muteins with Three Covalent and / or Non-Covalent Associations to Fully Connect the MonomersExample 6, SDS-PAGE and SEC Analysis of Mutant and Wild-Type LIGHT Interactions (Various Combinations of Covalent and / or Non-Covalent Associations)Multiple distinct LIGHT (TNFSF14) muteins were generated to assess their ability to form a polarized trimer and interact in a stable fashion.One mutein included mutations A: S182C / S200D / F202Y; B: S182D / S200C / G188C / F202Y; C: S182K / S200K / T191C / F202Y (BB 128). Another mutein included mutations A:S182C / S200D / F202Y; B: S200C / G188C / S182C / F202Y: C: T191C / S182K / S200C / F202Y (BB124). Another mutein included mutations A: S182C / F202Y; B: S200C / G188C / S182C / F202Y; C: T191C / S200C / F202Y (BB130).Another mutein included mutations A: S182C / S200C / F202Y; B:S200C / G188C / S182K / F202Y; C: T191C / S182C / S200D / F202Y (BB131). Another mutein included mutations A: S182C / S200D / F202Y; B: S200C / G188C / S182C / F202Y; C:T191C / S182R / S200C / F202Y(BB132). Another mutein included mutations A: S182C / G188C / S200D / F202Y; B: G188C / T191C / S200C / F202Y; C: S182K / T191C / F202Y (BB133).Another mutein included mutations A: S182C / G188C / S200D / F202Y; B: G188C / T191C / S200C / F202Y; C: S182R / T191C / F202Y (BB137). Another mutein included mutations A: S182C / G188C / S200D / F202Y; B: T191C / S200C / F202Y; C: S182K / F202Y (BB138). Another mutein included mutations A: S182C / G188C / S200D / F202Y; B: T191C / S200C / F202Y; C: S182R / F202Y (BB139).Docket No. BREACH-001 / WOOlAnother mutein included mutations A: S182K / F202Y; B: R124L / G188C / S200D / F202Y; C: R124L / T191C / V240D / F202Y (BB170). Another mutein included mutations A: S182C / F202Y; B: R124L / G188C / S200C / F202Y; C: R124L / T191C / V240D / F202Y (BB171). Another mutein included mutations A: S182R / F202Y; B: R124L / G188C / S200D / F202Y; C R124L / T191C / V240D / F202Y (BB172).Another mutein included mutations A: S182C / G188C / S200D / F202Y; B: S182F / T191C / S200D / F202Y; C: S182R / S200F / F202Y (BB175). Another mutein included mutations A: S182C / G188C / S200D / F202Y; B: S182H / T191C / S200C / F202Y; C: S 182R / S200H / F202Y (BB176). Another mutein included mutations A: S182C / S200D / F202Y; B: S182F / G188C / S200C / F202Y; C: S182R / T191C / S200F / F202Y (BB177).Another mutein included mutations A: S182C / S200D / F202Y; B:S182H / G188C / S200C / F202Y; C: S182R / T191C / S200H / F202Y (BB178). Another mutein included mutations A: S182C / G188C / F202Y; B: T191C / S200C / F202Y: C: F202Y (BB196).Another mutein included mutations A: F202Y; B: G188C / F202Y / D242C; C: N93C / T191C / F202Y (BB308). Another mutein included mutations A: F202Y; B: G188C / F202Y / I243C; C: N93C / T191C / F202Y: (BB309). Further, each of the monomers of both BB308 and BB309 include an additional C-terminus extension with protein sequence taken from the C-terminus of TNFSF11 (RANKL): RDID.The mutations were selected at positions in the association interface between LIGHT monomers and thus are likely to form disulfide bonds and salt bridges and increase stability of the oligomer. In the case of the V240D mutation, the aspartate at position 240 is designed to interact with the naturally occurring arginine at position 124. To avoid non-desired interactions and promote proper polarization, R124 is mutated into leucines in the relevant monomer.In agreement with improved stability conferred by the additional inter-chain interactions, many of these muteins exhibited an increase in expression yield, up to 5-fold increase compared to wild-type LIGHT trimers.Purity, stability, and expression yield of these muteins is in Table 15, below.Docket No. BREACH-001 / WOOlDocket No. BREACH-001 / WOOlTable 15: Properties of LIGHT Muteins with Various Combinations of Covalent and / or Non-Covalent Associations Between MonomersExample 7: Polarized LIGHT trimers maintain binding to LIGHT receptorsTo understand whether any mutations affected the ability of a LIGHT trimer to bind to its cognate receptors, a wildtype LIGHT trimer (BB118; Fig. 5A-D) and certain mutated LIGHT polarized trimers, including BB112, BB113, BB114, BB115 (Fig. 5 A), BB127, BB128 (Fig. 5B), BB132, BB133 (Fig. 5C), BB138, BB139, and BB174 (Fig. 5D) were tested in ELISA against human and mouse LIGHT receptors (HVEM-Fc and LTPR-Fc). Receptor-binding activity was assessed by comparing binding affinities of the mutant trimers to wild-type LIGHT. 96-well plate wells were coated with 2ug / mL of the LIGHT receptors and incubated with the wild type and mutant LIGHT trimers at concentrations ranging from OnM to lOOnM for Ihour at 25C. Following incubation with anti-His tag antibody, the binding of the LIGHT trimers to their receptors was detected by addition of TMB, and the reaction was stopped by the addition of IM HC1. The signal was read at 450nM and EC50 values were calculated using Graph Pad Prism 10. As seen in Fig. 5 and Table 16, below, the binding of the mutated LIGHT polarized trimers to human and mouse LIGHT receptors LTBR and HVEM was similar, or slightly reduced compared to that of wild-type LIGHT (BB 118). Specifically, the polarized LIGHT trimers with single new covalentDocket No. BREACH-001 / WOOl interactions (BB112, BB113, BB127) were most similar to wild-type, though other mutant trimers having multiple covalent and / or non-covalent interactions also showed good binding affinities. These results indicate that the individual mutations as well as the combinations of mutations tested do not affect significantly the binding of LIGHT to its receptors.Docket No. BREACH-001 / WOOlto their Cognate ReceptorsExample 8: Additional Mutations to Create New Non-C oval ent interactions and Stabilize TrimersIn some instances, certain sets of mutations in LIGHT trimers led to decreased or the absence of protein expression as seen with BB 122 (A: S182C / S20D, B: S182D / S200C, C: S182K / S200K) and BB123 (A: S182C / S200D, B: S182D / S200C / G188C, C:S182K / S200K / T191C). Without wishing to be bound by theory, the creation of new covalent and / or non-covalent associations within a trimeric ligand may cause the monomers within the ligand to bind closer and tighter than they would in a wildtype molecule. For example, a new covalent or non-covalent association forming between G188 and T191 in two monomers brings those monomers closer together, with the F202 residue of both monomers pointing towards the inside of the cavity. These residues may clash due to being brought tightly together. In these cases, addition of the F202Y mutation to BB122 and BB123 (resulting in BB126 and BB124, respectively) rescued the expression of these mutants, likely by the new tyrosine residues forming a lattice of hydrogen bonds (as shown in FIG. 2). As mentioned previously, addition of the F202Y mutation to BB113 (mutant BB127) improved yield, purity and melting temperature. These results indicate that the addition of the F202Y mutation to certain LIGHT trimers has a beneficial effect in line with its utility for therapeutic applications.In some instance, certain mutations in LIGHT can increase the stability of the disulfide bonds in order to promote the trimer polarization. FIG. 14 is a prediction model of trimeric ligands BB241 and BB127 obtained using the Chai-1 advanced open-source model, delivering AlphaFold3-level accuracy in predicting 3D structures of biomolecular complexes. As described herein, BB127 includes mutations A:F202Y, B:G188C / F202Y, C:T191C / F202Y, with a disulfide bond forming between G188C / T191C. BB241 uses BB127 as a scaffold (i.e., it includes the same mutations in each monomer) and also adds mutations A:R195D, B:S185E / R189D. As shown in FIG. 14, the addition of mutants S185E and R189D (in BB241 monomer B) interact with wild-type R195 (in BB241 monomer C) resulting in the positioning of R195 (BB241 monomer C) in such a way that it promotes the formation of a network of non-covalent interactions with the disulfide bond between G188C (monomer B) and T191(monomer C), resulting in improved trimer polarization, as compared to BB127. Mutation R195D in monomer A, prevents interaction between monomer A and E185 and DI 89 in monomer B, forcing monomers B and C to interact and form a disulfide bond between S188C and S191C.Docket No. BREACH-001 / WOOlExample 9: VHH-LIGHT FusionThis example illustrates the addition of functional moieties to trimeric ligands and explores whether these fusions interfere with the ability of LIGHT to bind to its cognate receptors. As shown in FIG. 6, VHH-LIGHT fusion molecules, e g., three monomer molecules each having a single domain antibody (VHH) fused to the N-terminus (BB49), were produced and compared to a trivalent VHH- Fc fusion protein including one or more VHH units fused to each Fc subunit (BB52). Binding of each fusion protein to its target was measured by SPR and the binding affinities were identical between the two fusion molecules tested.The VHH-LIGHT fusion protein’s binding to LIGHT receptors was measured by ELISA as described above. As shown in FIG. 6, the fusion of multiple VHH units to a LIGHT trimeric ligand did not affect the LIGHT trimeric ligand from binding to its receptors. Accordingly, a VHH-LIGHT fusion exhibit conserved VHH binding to its target, and maintained LIGHT’s functionality in terms of binding to its receptors.Example 10: Fv-fusion LIGHT Trimeric LigandThis example shows the obtention of a functional Fv-fused LIGHT trimeric ligand (FIGS. 7A- C). As shown in FIG. 7A, the variable domain of a Trastuzumab (monoclonal antibody that binds to HER2 protein) heavy chain (HC) was fused to the N-terminus of a LIGHT monomer, and the variable domain of the Trastuzumab light chain (LC) was fused to the C-terminus of the same LIGHT monomer. Expression of the protein resulted in a Trastuzumab Fv-LIGHT fusion homotrimer (BB97). As shown in FIG. 7B, the fusion protein conserved its ability to bind to HER2, while an anti-PDl Fv- LIGHT fusion (BB98) did not exhibit any binding to HER2. The Trastuzumab Fv-LIGHT fusion homotrimer conserved its ability to bind to LIGHT receptors (FIG. 7C). These results underscore that complex LIGHT fusions, both at the C-terminus and N-terminus of LIGHT, do not affect the ability of LIGHT to bind to its receptors, indicating that such fusions will maintain their functional and biological activities while also being endowed with new functional moieties.Example 11 : Design of multifunctional fusion proteins using polarized trimer scaffoldsIn this example, polarized LIGHT trimeric ligands (with mutations corresponding to BB133) were functionalized with various binding and functional moieties. As shown in FIG. 8A, a trimeric ligand comprising three monomers A, B, C, wherein monomer A was fused at its N-terminus to a VHH targeted against the tumor associated antigen (TAA) DLL3, and at its C-terminus to a VHH targeted against CD3, followed by a poly-histidine tag for purification. Monomer B was fused at its N-terminus to another identical anti-DLL3 VHH and at its C-terminus to monomer C. Monomer CDocket No. BREACH-001 / WOOl was fused at its C-terminus to a tumor targeting peptide. Thus, this fusion molecule has been engineered to localize to DLL3 (with two targeting moieties and an additional tumor-targeting peptide) and also includes an immune cell engager targeting CD3, representing a multifunctional and multispecific molecule.The fusion protein exhibited the expected molecular weight corresponding to a trimeric ligand (FIG. 8B, left; as seen on the SDS-PAGE gel at ~80kD), and forms predominantly one species corresponding to the expected protein, as shown on the SEC-HPLC micrograph (FIG. 8B, right). Accordingly, the fusion protein can be expressed and used for therapeutic purposes. Furthermore, the LIGHT fusion partners successfully conserved their biological function, as the fusion protein exhibited binding to both DLL3 (Fig. 8C) and CD3 (Fig. 8D) as determined by ELISA assays.FIGS. 9A-D illustrate another example of an engineered trimeric ligand with multiple functional moieties (BB183), using the polarized trimeric ligand BB133 as a scaffold. As shown in FIG. 9A, monomer A was fused at its N-terminus to a variable region of the heavy chain of an anti- PD-L1 antibody (CPI VH), and at its C-terminus to a half-life extension moiety (in this example, a VHH targeting human serum albumin), followed by a poly-histidine tag for purification. Monomer B was fused at its N-terminus to a variable region of the light chain of an anti-PD-Ll antibody (CPI VL) and at its C-terminus to monomer C. Monomer C was fused at its C-terminus to a tumor targeting peptide. Thus, this multifunctional fusion protein has been engineered to be a checkpoint inhibitor targeting PD-L1, while simultaneously targeting a tumor and also having an extended half-life to increase potency.As shown in FIG. 9B, the fusion protein exhibited the expected molecular weight as seen on the SDS-PAGE gel, and forms predominantly one species corresponding to the expected protein, as shown on the SEC-HPLC micrograph. Thus, this fusion protein has a good developability profile.The formation of a functional anti-PD-Ll binding domain, reflecting the proper interaction between the heavy chain variable domain (monomer A) with the light chain variable domain (monomer B) was tested by ELISA. The multifunctional molecule (BB 183) exhibited similar binding to mouse PD-L1 as compared to a corresponding full length anti-PD-Ll IgG (BB154), with EC50 of 8.7nM and 7.43 nM respectively. As a control, the polarized LIGHT trimer BB133 used as a scaffold in BB183 did not exhibit binding to PD-L1 (FIG. 9C).The presence and functionality of the half-life extension VHH was confirmed by ELISA as the protein bound to HSA with an EC50 of 7.4 nM. As a control the polarized LIGHT trimer BB133 used as a scaffold in BB183 did not exhibit binding to HSA (FIG. 9D).Docket No. BREACH-001 / WOOlThe ability of the multispecific fusion protein to bind to the LIGHT receptors mouse HVEM (FIG. 9E) and mouse LTBR (FIG. 9F) was tested by ELISA. The binding to the LIGHT receptors was not impaired by the addition of the fusion partners since the EC50 of BB183 binding to HVEM or LTBR was comparable to that of BB 133 (11.68nM vs 10.1 nM for HVEM, and 14.95nM vs 26.53nM for LTBR).FIGS. 10A-D illustrate another example of an engineered trimeric ligand (BB191) with multiple functional moieties. In this example, the trimeric ligand is a polarized trimeric ligand with mutations corresponding to BB127. The trimeric ligand was functionalized with various binders and functional moieties. Monomer A was fused at its N-terminus to a variable region of the heavy chain of an anti-PD-Ll antibody, and at its C-terminus to a poly-histidine tag. Monomer B was fused at its N-terminus to the variable region of the light chain of an anti-PD-Ll antibody and at its C-terminus to a tumor targeting peptide. Monomer C was fused at its C-terminus to a half-life extension VHH. Given that the anti-PD-Ll VH and VL were engineered to form a covalent interaction through introduction of cysteines, the fusion protein exhibited the expected molecular weight as seen on the SDS-PAGE, and forms predominantly one species corresponding to the expected protein, as shown on the SEC-HPLC micrograph. Furthermore, the formation of a functional anti-PD-Ll binding domain, reflecting the proper interaction between the heavy chain variable domain (monomer A) with the light chain variable domain (monomer B) was confirmed by ELISA as the protein bound to PD- L1 with an EC50 of 12.76 nM (comparable to the EC50 observed with the corresponding full-length IgG). The presence of a functional half-life extension VHH was confirmed by ELISA as the protein bound to HSA with an EC50 of 6.4 nM (FIG. 10D).Accordingly, these examples show that engineered trimeric ligands according to the disclosure can be successfully expressed and utilized to develop new therapeutic molecules.Example 12: Design of Multifunctional Fusion Proteins including PD-L1 and VEGF BindersFIGS. 11A-C illustrate additional examples of engineered trimeric ligands with a therapeutic utility. For example, a trimeric ligand can be fused to PD-L1 binders such as anti-PD-Ll VHHs or anti-PD-Ll Fv. In another example an engineered trimeric ligand can be fused to PD-L1 binders (such as anti-PD-Ll VHH or anti-PD-Ll Fv), and to VEGF binders (such as anti-VEGF VHH). The engineered trimeric ligand can also be fused to a half-life extension moiety such as an anti-HSA VHH and to tumor targeting / tumor homing moieties such as the iRGD peptide. Specifically, FIG. 11A depicts a trimeric ligand with two PD-L1 binding moieties, a half-life extension moiety, and an iRGD peptide. Such a trimeric ligand can bind to multiple PD-L1 proteins (increasing the activation andDocket No. BREACH-001 / WOOl function of T cells) while simultaneously targeting a tumor and showing increased half-life. FIG. 1 IB depicts the trimeric ligand of FIG. 11 A with an additional VEGF binding moiety; this trimeric ligand will have the same functionality (binding to PD-L1 and targeting tumors), and will also bind to VEGF proteins, thus reducing tumor growth by inhibiting angiogenesis. FIG. 11C depicts a similar trimeric ligand having two VEGF binders and one PD-L1 binder, thus modulating its affinity for VEGF over PD-L1.The rationale for using anti angiogenic drugs as partners for immune checkpoint inhibitors such as anti-PD-1 and anti-PD-Ll antibodies, relies primarily on blocking the multiple immunosuppressive effects of VEGF and inducing several different vascular-modulating effects that can stimulate immunity, such as vascular normalization leading to increased intratumoural blood perfusion and flow, and inhibition of pro-apoptotic effects of endothelial cells on T cells, among others. Recently, the combination of VEGF blockers with checkpoint inhibitors was achieved in the form of single agents, i.e., bispecific antibodies. One such compound, AK112 exhibited very promising clinical activity in patients with advanced non-small cell lung cancer (AK112, a novel PD- 1 / VEGF bispecific antibody, in combination with chemotherapy in patients with advanced non-small cell lung cancer (NSCLC): an open-label, multicenter, phase II trial; Zhao, Yuanyuan et al. eClinicalMedicine, Volume 62, 102106, the contents of which are hereby incorporated by reference in their entirety) confirming the merit of that bispecific approach. See also Tzuri et al., Developing a dual VEGF / PDL1 inhibitor based on high-affinity scFv heterodimers as an anti-cancer therapeutic strategy, Sci Rep. 2023 Jul 24; 13:11923, the contents of which are incorporated by reference in their entirety.The inventor has recognized and appreciated that LIGHT’ s innate functions could complement and improve PD-L1 / VEGF targeting through several different mechanisms of action, including but not limited to: (1) Immune stimulation: Activation of T cells through HVEM (enhancing cytotoxicity); promotion of DC maturation and function; and increase in infiltration of immune cells into tumors. (2) Vascular effects: Normalization of the tumor vasculature through synergy with VEGF blockade; and potential to improve drug delivery. (3) Stromal remodeling: Reduction of tumor fibrosis; Improvement of immune cell access; potential enhancement of therapeutic antibody penetration. Accordingly, anti-PD-Ll and anti-VEGF LIGHT trimeric ligands as described herein (e.g., FIGS. 9A- F, FIGS. 10A-D, FIGS. 11A-F) have significant potential to outperform bispecific antibodies in terms of therapeutic ability.Docket No. BREACH-001 / WOOlAs previously noted, any suitable PD-L1 binder may be incorporated into embodiments of the disclosure, including VHH and Fv. Examples of suitable anti-PD-Ll VHH sequences can be found at least in Kang-Pettinger et al., “Identification, binding, and structural characterization of single domain anti-PD-Ll antibodies inhibitory of immune regulatory proteins PD-1 and CD80”, J Biol Chem. 299(1): 102769 (2022), the contents of which are hereby incorporated by reference. Several anti-PD- Ll VHH sequences are in Table 17, below.Table 17: Exemplary VHH Sequences Targeting PD-L1Example 13: Multifunctional Molecules as T cell EngagersThe efficacy of T cell engagers (such as anti -CD3 -based binders) is limited by the immunosuppressive tumor microenvironment and poor T cell infiltration and function. These limitations can be addressed by incorporating T cell engagers into a TNFSF trimeric ligand as described herein, such as in Example 11 and FIGS 8A-B.FIG. 1 IE depicts an engineered LIGHT trimeric ligand that includes an anti-CD3 moiety (anti- CD3 VHH), two anti-DLL3 moieties (each an anti-DLL3 VHH), a tumor targeting moiety (iRGD peptide), and a half-life extension moiety (anti-HSA VHH). FIG. 1 IF depicts a similar trimeric ligand where the two anti-DLL3 moieties have been replaced with a single anti-DLL3 Fv (VH / VL). Functionally, these trimeric ligands mimic a bispecific T-cell engager (BiTE) by recruiting T cells to DLL3 -expressing tumor cells (such as in small cell lung cancer, SCLC) and promoting T cell- mediated killing. However, the engineered LIGHT trimeric ligands offer multiple advantages over BiTEs by enhancing T cell activation, tumor infiltration, and response durability.First, LIGHT enhances T cell function by delivering co-stimulatory signals beyond CD3 engagement via HVEM, supporting T cell survival, persistence, and cytokine production at the tumor site. Second, LIGHT improves T cell infiltration by modulating tumor vasculature and chemokine production. This facilitates T cell migration into the tumor, increasing the pool of T cells available for engagement and enhancing immune accessibility to tumor sites. Third, LIGHT provides synergisticDocket No. BREACH-001 / WOOl activation, integrating co-stimulatory signaling with tumor-specific targeting (e.g., DLL3) and T cell activation (CD3). These combined signals enhance T cell proliferation and cytotoxic function, potentially leading to more efficient tumor eradication than traditional T cell engagers alone.LIGHT also modifies the tumor microenvironment, helping to counteract local immunosuppression that often limit T cell access and function. By sustaining T cell activity and preventing rapid exhaustion, LIGHT may also support memory T cell development, contributing to longer-lasting immune responses. Thus, a LIGHT -based trimeric ligand not only addresses the inherent limitations of CD3 T cell engagers, but also enhances tumor infiltration, immune activation, and response durability, ultimately improving the efficacy of T cell-mediated cancer therapies.A LIGHT-based trimeric ligand may also have a protective effect specifically in SCLC, where LIGHT expression is significantly down-regulated. LIGHT expression is associated with a better prognosis. See Zhang et al., Tumor Necrosis Factor Family Member Profile Predicts Prognosis and Adjuvant Chemotherapy Benefit for Patients with Small-Cell Lung Cancer, Front Immunol. 2021 Nov 18: 12:745769, the contents of which are hereby incorporated by reference in their entirety.Such a LIGHT / DLL3 / CD3 multispecific protein can be tested in cell-based assay of T cell- redirected cytotoxicity to assess its utility as a T cell engager. For that, frozen human PBMCs are thawed and resuspended with RPMI1640 complete medium, incubated in 37°C 5% CO2 incubator overnight. PBMCs are then resuspended with RPMI1640 medium (with 2% HLFBS) at density of 2E6 cells / mL. 2E5 PBMCs (lOOpL) are dispensed into each well of 96-well round bottom plate. The target cells, SHP-77 that express DLL3 are thawed and resuspended in RPMI1640 medium (with 2% HLFBS) at density of 4E5 cells / mL. 2E4 tumor cells (50pl) (E:T=10:l) are dispensed into each well of 96-well round bottom plate. 50pL of 4x test articles or isotype control are added into the wells and incubated for 24h. After 24h incubation, cell cytotoxicity was detected using an LDH assay. For example, as shown in FIG. 11G two fusion proteins were compared, BB283 comprising an anti-DLL3 Fv (with the light chain and heavy chain variable regions, each fused to the N-ter of a distinct LIGHT (BB127) monomer); and BB311 comprising an anti-DLL3 VHH fused to the N-ter of a LIGHT (BB 127) monomer. As shown, each of these LIGHT / DLL3 / CD3 multispecific proteins in the presence of PBMCs and DLL3 -expressing SHP-77 target cells lead to dose-dependent cytotoxicity of the target cells.Example 14: Multifunctional Molecules can Activate T cells in the Presence of Cancer CellsThe efficacy of checkpoint inhibitors, such as anti-PD-Ll binders, is limited by poor T cell tumor infiltration and function. This efficacy can be improved by combining anti-PD-Ll binders intoDocket No. BREACH-001 / WOOl a TNFSF trimeric ligand as described herein. For example, FIG. 11D depicts an engineered LIGHT trimeric ligand that incorporates an Anti-PD-Ll Fv (VH / VL), a tumor targeting moiety (iRGD peptide), and a half-life extension moiety (anti-HSA VHH). This trimeric ligand offers significant advantages over IgG-based formats by addressing multiple limitations of checkpoint inhibitors.First, LIGHT modifies the tumor microenvironment by promoting vascular changes that enhance T cell infiltration and by inducing chemokines (e.g., CXCL9 / 10) to attract T cells. This reduces immunosuppressive cell populations and creates an inflammatory microenvironment conducive to anti-tumor immune responses. Second, LIGHT provides co-stimulatory signals through HVEM, enhancing T cell activation, proliferation, and memory T-cell development while reducing exhaustion. Third, these mechanisms complement and synergize with the anti-PD-Ll domain by increasing T cell availability and functionality, improving tumor antigen presentation and recognition, and sustaining immune responses across multiple pathways.Finally, LIGHT directly impacts cancer cells by activating lymphotoxin 0 receptor signaling, disrupting tumor survival signals, and sensitizing tumors to immune-mediated killing. Together, these effects enable a LIGHT-based trimeric ligand to overcome the limitations of anti-PD-Ll therapy in “cold” tumors by driving complementary anti-tumor mechanisms and addressing resistance to checkpoint inhibition, ultimately leading to more durable responses.Such a LIGHT / PD-L1 fusion protein can be tested in cell-based assay to evaluate the activation of T cells in the presence of cancer cells, measured by the detection of IFN-gamma secretion. For that, frozen mouse PBMCs or T cells are thawed and resuspended with RPMI1640 complete medium, incubated in 37 °C 5% CO2 incubator overnight. PBMCs or T cells are then resuspended with RPMI1640 medium (with 2% HI-FBS) at density of 2E6 cells / mL. 2E5 PBMCs(lOOpL) are dispensed into each well of 96-well round bottom plate. The target cells, MC-38 cells are thawed and resuspended in RPMI1640 medium (with 2% HI-FBS) at density of 4E5 cells / mL. 2E4 tumor cells (50pl) (E:T=10: l) are dispensed into each well of 96-well round bottom plate. 50pL of4x test articles or isotype control are added into the wells and incubated for 24h. After 24h incubation, the supernatant is collected, and INF-gamma levels are measured. The ability of the LIGHT / PD-L1 multispecific protein to induce IFN-gamma secretion can be compared to established anti-PD-Ll antibodies, such as the 10F.9G2 monoclonal antibody and similar antibodies. FIG. 12 shows the results of an ELISA assay where T cell-secreted IFN-gamma levels are measured. In this experiment, as described above, mouse PBMC were incubated for 24 hours with MC-38 in the presence or absence of an isotype control antibody, the 10F.9G2 anti-mouse PD-L1 monoclonal antibody, or the LIGHT / PD-L1Docket No. BREACH-001 AVOOl multispecific protein that includes the Fv of 10F.9G2 and an anti-HSA VHH acting as a HLE. The polarized trimer used for this protein is BB127. Lower concentrations of the LIGHT / PD-L1 multispecific protein leads to IFN-y secretion as compared to the parent anti-PD-Ll IgG antibody 10F.9G2; and the maximum response elicited by the LIGHT / PD-L1 multispecific protein is larger than that of the parent anti-PD-Ll IgG antibody 10F.9G2. Altogether, these results indicate that the LIGHT / PD-L1 multispecific protein leads to enhanced in vitro T cell activation in the presence of MC38 cells, compared to the corresponding parent IgG antibody.Example 15: Design and expression of a polarized CD40L trimeric ligandCD40L polarized trimer muteins were designed following a similar approach as for the LIGHT polarized trimer mutein designs. Residues from two different monomers that do not interact but are in close proximity were mutated to cysteines to enable the formation of one inter-chain disulfide bond. Wildtype and sCD40L muteins were expressed and the formation of a covalent dimer was assessed by SDS-PAGE, and confirmed by the presence of a band corresponding to a dimeric species, and a band corresponding to a monomeric species (FIG. 13A-D). The mutein included mutations B: T211C; C: G219C (BB193); or C: S213C (BB438). and the removal of a free Cysteine (BB443), and the removal of a free cysteine and an intrachain disulfide bond (BB482 and BB490). Furthermore, the increased stability of the trimer was reflected by a higher Tm of BB193 (69.64C) compared to that of wildtype CD40L, BB136 (63.32C).Docket No. BREACH-001 / WOOlTable 18: Properties of CD40L Muteins with Covalent Associations Between MonomersExample 16: Design and expression of a polarized 4-1BBL trimeric ligand4-1BBL polarized trimer muteins were designed following a similar approach as for the LIGHT polarized trimer mutein designs. Residues from two different monomers that do not interact but are in close proximity were mutated to cysteines to enable the formation of one inter-chain disulfide bond. Wildtype and a 4-1BBL mutein were expressed, and the formation of a covalent dimer was assessed by SDS-PAGE, and confirmed by the presence of a band corresponding to a dimeric species, and a band corresponding to a monomeric species (FIG. 13E). The mutein included mutations A: D184C; B: R193C (BB400). Furthermore, the increased stability of the trimer was reflected by a higher Tm of BB400 (84.15C) compared to that of wildtype 4-1BBL, BB399 (58.39C). Furthermore,Docket No. BREACH-001 / WO01As seen in FIG. 14 and Table 19 below, the binding of the mutated 4-1BBL polarized trimers to human 4- IBB was slightly improved.Table 19: Properties of the 4-1BBL Mutein with Covalent Associations Between MonomersExample 17: Design and expression of a polarized TRAIL trimeric ligand TRAIL polarized trimer muteins were designed following a similar approach as for the LIGHT polarized trimer mutein designs, with the addition of using the naturally occurring cysteine 230 to create additional disulfide bonds. Residues from two different monomers that do not interact but are in close proximity were mutated to cysteines to enable the formation of one inter-chain disulfide bond. In parallel, the cysteine 230 in one monomer was mutated into a histidine, a serine, or an alanine, to enable the targeted formation of a disulfide bond between the two intact cysteines. Specifically, the inventor has recognized and appreciated that cysteine 230 is present at the center of the interface between the monomers in the trimeric ligand. The cysteines naturally coordinate with a zinc atom. However, when expressed in a laboratory setting, undesired disulfide bonds are formed, resulting in an excess of dimers. Mutating one of the monomers at position 230 to (e g.) a histidine, serine, or alanine residue will prevent an undesired disulfide bond while not interfering with interaction with zinc. Exemplary muteins can include mutations A: D203C; B: C230H / S232C (BB491); A: D203C; B: C230S / S232C (BB514); and A: D203C; B: C230A / S232C (BB515).Docket No. BREACH-001 / WO01Table 20Example 18: Evaluation of an anti-PD-Ll / anti -Human serum albumin / LIGHT (BB127) fusion efficacy in subcutaneous MC38 tumor model in C57BL / 6-hALB / hFCRN mice.FIG. 15A is a cartoon depicting the structure of an exemplary trimeric ligand (BB363), a polarized LIGHT trimer (BB127) fused to two anti-mouse PD-L1 VHH (fused to the N-ter of monomers A and C), and to one anti-human serum albumin VHH (fused to the C-ter of monomer B). This example evaluates the efficacy of BB363 in a mouse tumor model.C57BL / 6-hALB / hFcRn,(male and female) mice, aged 6-8 weeks, were subcutaneously inoculated with MC38 cells (1 * 106 cells per mouse, in PBS) on day 0. On day 6 post inoculation, based on the tumor volume (~80 mm3), 16 mice (8 females and 8 males) were enrolled and randomized into two groups (4 females and 4 males per group): Group 1 (vehicle, intraperitoneally, twice per week for 6 doses), Group 2 (BB363, 7.5 mg / kg, intraperitoneally, twice per week for 6 doses), The dosing began on the day of grouping (day 0 post grouping and dosing). Animal weight and tumor volume were measured twice per week (FIG. 15B). Two mice in Group 1 were terminated early when their tumor volume reached more than 3000 mm3. On day 17, (all animals alive), the average body weight of Group 1 and 2 was 22.91±1.09g, and 21.59±0.91g respectively. On day 25, all remaining mice were terminated. The average body weight of Groups 1 and 2 was 26.10±1.25 g, and 23.48±0.79g respectively.Docket No. BREACH-001 / WOOlOn day 25, the tumor volume of Group 1 (the control group) was 4124.73±391.23 mm3. The tumor volume of Group 2 was 1521.42±354.24 mm3. The TGI of Group 2 was 63.12% compared with Group 1 and showed a statistically significant difference (P < 0.001). The average tumor weight of Groups 1 and 2 was 3.7424±0.2955g and 1.4556±0.3901g respectively. These results indicate that a fusion protein consisting of a LIGHT polarized trimer (BB 127), and two distinct VHHs (anti-mouse PD-L1 and anti-human serum albumin) can not only be produced, but it can lead to anti -turn or activity in vivo.While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Docket No. BREACH-001 / WOOlCLAIMS1. A multifunctional molecule, comprising a trimeric ligand with three monomer molecules, wherein:(a) each monomer molecule is selected from a tumor necrosis factor superfamily (TNFSF) member, a TNFSF -like member, or a combination thereof;(b) at least one monomer molecule comprises a modification at one or more amino acid positions that forms one or more new covalent and / or non-covalent associations between at least two of the monomers; and(c) at least one functional moiety is fused to either the N-terminus or C-terminus of one of the monomer molecules.

2. The multifunctional molecule of claim 1, further wherein: (d) one of the monomer molecules further comprises a C-terminal extension, wherein the C-terminal extension forms a new covalent and / or non-covalent association between at least two of the monomers.

3. The multifunctional molecule of claim 2, wherein the C-terminal extension sequence comprises X1X2X3X4, X1X2X3, or X1X2, wherein X2 or X3 comprise a cysteine residue.

4. The multifunctional molecule of claim 3, wherein the C-terminal extension comprises an amino acid sequence of either RCID or RDCD.

5. The multifunctional molecule of any prior claim, wherein one or more naturally occurring cysteine residues within one or more of the monomer molecules are replaced with serine or histidine residues.

6. The multifunctional molecule of any prior claim, wherein the trimeric ligand comprises a first pair of complementary amino acid modifications, the one or more pairs comprising a first pair comprising an amino acid modification in a first monomer molecule and a corresponding amino acid modification in a second monomer molecule, wherein the amino acid modifications form a new covalent and / or non-covalent association between the monomers.

7. The multifunctional molecule of claim 6, wherein the trimeric ligand comprises a second pair of complementary amino acid modifications, wherein the second pair comprises a second amino acid modification in the first monomer molecule and a corresponding amino acid modification in a third monomer molecule, wherein the amino acid modifications form a new covalent and / or non- covalent associations between the monomers.Docket No. BREACH-001 / WOOl8. The multifunctional molecule of any prior claim, wherein the trimeric ligand comprises one or more pairs of complementary amino acid modifications, each pair forming a new covalent and / or non-covalent association between the monomers.

9. The multifunctional molecule of any prior claim, wherein the new covalent or non-covalent associations cause the three monomer molecules to associate in a specific configuration to create the trimeric ligand.

10. The multifunctional molecule of any prior claim, wherein a pair of complementary amino acid modifications comprises a modification at an amino acid position in a first monomer and a modification at an amino acid position in a second monomer, wherein the atoms in the amino acid positions in the first and second monomers that engage in interchain interaction within the trimeric ligand are between 2-6 angstroms from one another.

11. The multifunctional molecule of any prior claim, wherein the covalent associations comprise disulfide bonds created by new cysteine residues introduced into the at least two monomers.

12. The multifunctional molecule of any prior claim, wherein the non-covalent associations comprise salt bridges, hydrogen bonds, and / or hydrophobic interactions created by new residues introduced into at least one monomer.

13. The multifunctional molecule of any prior claim, wherein the trimeric ligand retains its ability to bind to its cognate receptors.

14. The multifunctional molecule of any prior claim, wherein the monomer molecules are TNFSF members, and are selected from the group consisting of: TNF-alpha, LT-alpha, LT-beta, OX40L, CD40L, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, TL1A, GITRL, EDA-A1, and EDA-A2.

15. The multifunctional molecule of claim 14, wherein the monomer molecules comprise LIGHT, and the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: N93, R124, S182, Q183, S185, G188, R189, T191, R195, S200, R232, and V240, as compared to wild-type LIGHT.

16. The multifunctional molecule of claim 15, wherein one of the monomer molecules further comprises an additional C-terminal extension sequence of R241X242X243D244, X241X242X243X244, or X241X242X243, wherein X242 orX243 comprise a cysteine residue.Docket No. BREACH-001 / WOOl17. The multifunctional molecule of claim 15 or claim 16, wherein the modifications that form a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: N93C, R124A, R124L, S182C, S182D, S182F, S182H, S182K, S182R, S182W, Q183R, S185E, G188C, T191C, R195D, S200A, S200C, S200D, S200F, S200H, S200K, S200W, R232D, V240D, X242C, and X243C, as compared to wild-type LIGHT.

18. The multifunctional molecule of any one of claims 14-18, wherein the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: 93 / 242; 93 / 243; 182 / 200; 183 / 232; 188 / 191; 189 / 185; 185 / 195; and 124 / 240, as compared to wild-type LIGHT.

19. The multifunctional molecule of any one of claims 14-18, wherein the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: N93C / 242C; N93C / 243C; S182C / S200C; G188C / T191C; R124 / V240D; S182R / S200D; S182K / S200D; S182F / S200F; S182H / S200H; Q183R / R232D; R189D / R195; and S185E / R195, as compared to wild-type LIGHT.

20. The multifunctional molecule of any one of claims 14-19, wherein one, two, or three of the monomer molecules further comprise one or more amino acid modifications selected from the group consisting of: R124A, R124L, Y142H, Y144H, S182A, S185E, R189D, R195D, S200A, F202Y, or F238Y, as compared to wild-type LIGHT.

21. The multifunctional molecule of claim 14, wherein the monomer molecules comprise CD27L, and the modification that forms a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: SI 17 and Q149, as compared to wild-type CD27L.

22. The multifunctional molecule of claim 16, wherein the modifications that form a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: SI 17C and Q149C, as compared to wild-type CD27L.

23. The multifunctional molecule of claim 16 or claim 17, wherein the new covalent and / or non- covalent association formed between the monomers comprises one or more of the following paired amino acid modifications in at least two monomers: 117 / 149, as compared to wild-type CD27L.

24. The multifunctional molecule of claim 14, wherein the monomer molecules comprise CD40L, and the modification that forms a new covalent or non-covalent association between theDocket No. BREACH-001 / WOOl monomers comprise a modification at one or more amino acid positions selected from the group consisting of: A208, T211, S213, G219, and S222, as compared to wild-type CD40L.

25. The multifunctional molecule of claim 24, wherein the modifications that form a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: A208C, A208D, T211C, G219C, S213C, S222R, S222C, and S222K, as compared to wild-type CD40L.

26. The multifunctional molecule of claim 24 or claim 25, wherein the new covalent and / or non- covalent association formed between the monomers comprises one or more of the following paired amino acid modifications in at least two monomers: 208 / 222; 211 / 219; and 211 / 213, as compared to wild-type CD40L.

27. The multifunctional molecule of any one of claims 22-26, wherein the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: A208D / S222R; A208C / S222C;A208D / S222K; T211C / G219C; and T211 / S213C, as compared to wild-type CD40L.

28. The multifunctional molecule of any one of claims 22-27, wherein one or more of the monomers comprises one more amino acid modifications that substitute a naturally occurring cysteine residue with a serine residue, wherein the modifications comprise: C194S and / or C178S.

29. The multifunctional molecule of claim 14, wherein the monomer molecules comprise TL1A, and the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of R96, DI 46, Ml 97, and D242, as compared to wild-type TL1A.

30. The multifunctional molecule of claim 29, wherein the modifications that form a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: R96D, D146R, D146K, D146N, M197C, and D242C as compared to wild-type TL1A.

31. The multifunctional molecule of claim 29 or claim 30, wherein the new covalent and / or non- covalent association formed between the monomers comprises one or more of the following paired amino acid positions in at least two monomers: 197 / 242; and 146 / 96, as compared to wild-type TL1A.

32. The multifunctional molecule of any one of claims 29-31, wherein the new covalent and / or non-covalent association formed between the monomers comprises one or more of the followingDocket No. BREACH-001 / WOOl pairs of amino acid positions in at least two monomers: M197C / D242C; R96D / D146R; and M197C / D242C, as compared to wild-type TL1A.

33. The multifunctional molecule of any one of claims 29-32, wherein one, two, or three of the monomer molecules further comprise one more amino acid modifications that substitute a naturally occurring cysteine residue with a serine residue, wherein the modifications comprise: C163S and / or C203S, as compared to wild-type TL1A.

34. The multifunctional molecule of any one of claims 29-33, wherein one, two, or three of the monomer molecules further comprise one or more amino acid modifications selected from the group consisting of: F148Y, and F244Y, as compared to wild-type TL1A.

35. The multifunctional molecule of claim 14, wherein the monomer molecules comprise LTB, and the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: L138, G181, and S204, as compared to wild-type LTB.

36. The multifunctional molecule of claim 35, wherein the modifications that form a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: L138K, L138R, G181C, G181D, and S204C, as compared to wild-type LTB.

37. The multifunctional molecule of claim 35 or claim 36, wherein the new covalent and / or non- covalent association formed between the monomers comprises one or more of the following paired amino acid positions in at least two monomers: 181 / 204; and 138 / 181, as compared to wild-type LTB.

38. The multifunctional molecule of any one of claims 35-37, wherein the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: L138R / G181D; L138K / G181D; and G181C / S204C, as compared to wild-type LTB.

39. The multifunctional molecule of claim 14, wherein the trimeric ligand is a heterotrimeric LTA-LTB-LTB complex, and the modification that forms a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: LTA: M154 and L164, as compared to wild-type LTA; and LTB: R160, G191, A192, Y212, and S214, as compared to wild-type LTB.

40. The multifunctional molecule of claim 39, wherein the modifications that form a new covalent or non-covalent association between the monomers comprise a modification at one or moreDocket No. BREACH-001 / WOOl amino acid positions selected from the group consisting of: LTA: L164E, M154C, as compared to wild-type LTA; and LTB: R160E, A192C, G191C, Y212C, S214C, as compared to wild-type LTB.

41. The multifunctional molecule of claim 39 or claim 40, wherein the new covalent and / or non- covalent association formed between the monomers comprises one or more of the following paired amino acid positions in at least two monomers: LTA: 154 / LTB:212; LTB: 192 / LTB:212; LTB: 191 / LTB: S214, as compared to wild-type LTA and wild-type LTB.

42. The multifunctional molecule of any one of claims 38-41, wherein the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: LTA:M154C / LTB:Y212C;LTB:A192C / LTB:Y212C; LTB:G191C / LTB:S214C, as compared to wild-type LTA and wild-type LTB.

43. The multifunctional molecule of claim 14, wherein the monomer molecules comprise 4- 1BBL, and the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: G80, D184, R193, and T241C, as compared to wild-type 4-1BBL.

44. The multifunctional molecule of claim 43, wherein the modifications that form a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: G80C, D184C, R193C, and T241C, as compared to wild-type 4-1BBL.

45. The multifunctional molecule of claim 43 or claim 44, wherein the new covalent and / or non- covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: 184 / 193; and 80 / 241, as compared to wild-type 4- 1BBL.

46. The multifunctional molecule of any one of claims 43-46, wherein the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: D184C / R193C; and G80C / T241C; as compared to wild-type 4-1BBL.

47. The multifunctional molecule of claim 14, wherein the monomer molecules comprise TRAIL, and the modification that forms a new covalent or non-covalent association between the monomers comprises a modification at one or more amino acid positions selected from the group consisting of: D203, and S232, as compared to wild-type TRAIL.Docket No. BREACH-001 / WOOl48. The multifunctional molecule of claim 47, wherein the modifications that form a new covalent or non-covalent association between the monomers comprise a modification at one or more amino acid positions selected from the group consisting of: D203C and S232C, as compared to wild-type TRAIL.

49. The multifunctional molecule of claim 47 or claim 48, wherein the new covalent and / or non- covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: 203 / 232, as compared to wild-type TRAIL.

50. The multifunctional molecule of any one of claims 47-49, wherein the new covalent and / or non-covalent association formed between the monomers comprises one or more of the following pairs of amino acid positions in at least two monomers: D203C / S232C, as compared to wild-type TRAIL.

51. The multifunctional molecule of any one of claims 47-50, wherein one, two, or three of the monomer molecules further comprise one or more amino acid modifications selected from the group consisting of: C230H, C230S, and C230A.

52. The multifunctional molecule of any prior claim, wherein the trimeric ligand further comprises a modification at one or more amino acid positions to remove unpaired cysteine residues.

53. The multifunctional molecule of any prior claim, wherein the trimeric ligand comprises two, three, four, five, or six functional moieties, wherein each functional moiety is fused to either the N- terminus or C-terminus of one of the monomer molecules.

54. The multifunctional molecule of claim 53, wherein the functional moieties comprise:(i) a tumor-targeting moiety that binds to a cancer antigen; and one, two, three, four, or five of:(ii) an immune cell engager;(iii) a cytokine molecule;(iv) a stromal modifying moiety;(v) a checkpoint inhibitor;(vi) an enzyme; and(vii) a half-life extension moiety.

55. The multifunctional molecule of claim 54, wherein: if (ii) and (iii) are absent, then (i) and (iv) are present, if one (i) and one (ii) are present, then (iii) or (iv) or both are present, and if one (i) and one (iii) are present, then (ii) or (iv) or both are present.Docket No. BREACH-001 / WOOl56. The multifunctional molecule of any one of claims 53-55, wherein the trimeric ligand comprises at least one tumor-targeting moiety that binds to a cancer antigen.

57. The multifunctional molecule of any one of claims 53-56, wherein the trimeric ligand comprises at least two tumor targeting moieties that bind to a cancer antigen.

58. The multifunctional molecule of any one of claims 53-57, wherein the trimeric ligand comprises at least three tumor targeting moieties that bind to a cancer antigen.

59. The multifunctional molecule of claim 57 or claim 58, wherein the tumor targeting moieties bind to different cancer antigens.

60. The multifunctional molecule of any one of claims 53-59, wherein the tumor-targeting moiety localizes the trimeric ligand to the cancer, thereby activating the immune system against the cancer.

61. The multifunctional molecule of any one of claims 53-60, wherein the cancer antigen is selected from the group consisting of: Delta-like protein 3 (DLL3), B-cell maturation antigen (BCMA), carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (Her2), epithelial cell adhesion molecule (EpCAM), CD20, CD26, CD123, CD30, CD33, CD47, CD52, CD133, glycoprotein A33 (gpA33), mucins, tumor associated glycoprotein-72 (TAG-72), type IX collagen (CIX), glutamate carboxypeptidase II (PSMA), folate-binding protein, GD2, GD3, GM2, vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), integrin, aV03, a5 1, ERBB2, ERBB3, mesenchymal epithelial transition (MET), B7-H3, insulin-like growth factor-I receptor (IGFIR), ephrin type-A receptor 3 (EPHA3), TRAIL receptor 1 (TRAILR1), TRAIL receptor 2 (TRAILR2), receptor activator of nuclear factor kappa beta (RANKL), fibroblast activation protein (FAP), claudin 18.2, mesothelin, receptor tyrosine kinase like orphan receptor 1 (ROR1), epidermal growth factor receptor variant III (EGFR VIII), six-transmembrane epithelial antigen of the prostate- 1 (STEAP1), six-transmembrane epithelial antigen of the prostate-2 (STEAP2), orphan G protein- coupled receptor, class C group 5 member D (GPRC5D), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), and tenascin.

62. The multifunctional molecule of any one of claims 53-61, wherein the trimeric ligand comprises at least one immune cell engager.

63. The multifunctional molecule of any one of claims 53-62, wherein the trimeric ligand comprises at least two immune cell engagers.Docket No. BREACH-001 / WOOl64. The multifunctional molecule of any one of claims 53-63, wherein the immune cell engager comprises an immune checkpoint inhibitor, a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, a myeloid cell engager, and / or a Toll-Like Receptor (TLR).

65. The multifunctional molecule of any one of claims 53-64, wherein the trimeric ligand comprises an immune checkpoint inhibitor.

66. The multifunctional molecule of any one of claims 53-65, wherein the immune checkpoint inhibitor is selected from the group consisting of: PD-1, PD-L1, and CTLA-4.

67. The multifunctional molecule of any one of claims 53-66, wherein the trimeric ligand comprises a stromal modifying moiety.

68. The multifunctional molecule of claim 67, wherein the stromal modifying moiety is selected from the group consisting of: a vascular modifying moiety (VEGF), Fibroblast association protein (FAP), and extracellular matrix binding protein.

69. The multifunctional molecule of claim 53 or 54, comprising a first tumor targeting moiety targeting PD-L1, a second tumor targeting moiety comprising a tumor targeting peptide, and a halflife extension moiety.

70. The multifunctional molecule of claim 69, wherein the first tumor targeting moiety comprises an anti-PD-Ll VHH fused to at least one monomer.

71. The multifunctional molecule of claim 70, wherein the anti-PD-Ll VHH comprises a VHH sequence selected from one of SEQ ID NOs: 27-32.

72. The multifunctional molecule of claim 69, wherein the first tumor targeting moiety comprises an anti-PD-Ll Fv comprising a VH and a VL, wherein the VH and VL are each fused to at least one monomer.

73. The multifunctional molecule of any one of claims 69-72, further comprising a third tumor targeting moiety comprising a VEGF targeting moiety.

74. The multifunctional molecule of claim 53 or 54, comprising a first tumor-targeting moiety targeting DLL3, a second tumor targeting moiety comprising a tumor targeting peptide, a half-life extension moiety, and an immune cell engager.

75. The multifunctional molecule of claim 74, wherein the first tumor-targeting moiety comprises an anti-DLL3 VHH fused to at least one monomer.

76. The multifunctional molecule of claim 74, wherein the first tumor-targeting moiety comprises an anti-DLL3 Fv comprising a VH and a VL, wherein the VH and VL are each fused to at least one monomer.Docket No. BREACH-001 / WOOl77. The multifunctional molecule of any one of claims 74-76, wherein the immune cell engager targets CD3.

78. The multifunctional molecule of claim 77, wherein the immune cell engager comprises an anti-CD3 VHH.

79. The multifunctional molecule of any one of claims 69-78, wherein the half-life extension moiety comprises a human serum albumin (HSA) binding protein.

80. The multifunctional molecule of any one of claims 69-79, wherein the trimeric ligand is LIGHT.

81. The multifunctional molecule of any prior claim, wherein the multifunctional molecule promotes T cells, Myeloid cells, Dendritic cells, or cytokine immunity against target cells, e.g., cancer.

82. A pharmaceutical composition comprising (a) the multifunctional molecule of any prior claim, and (b) a pharmaceutically acceptable carrier.

83. One or more nucleic acids encoding the multifunctional molecule of any one of claims 1-81.

84. An expression vector comprising the one or more nucleic acids of claim 83.

85. A host cell comprising the one or more nucleic acids of claim 83 or the expression vector of claim 85.

86. A method of making a multifunctional molecule comprising culturing the host cell of claim 85 and recovering the multifunctional molecule from the cell culture.

87. A method of treating a disease or condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the multifunctional molecule of any one of claims 1-81.

88. The method of claim 87, wherein the disease or condition comprises a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease or a host-versus-graft disease.

89. A method of treating cancer in a patient, comprising administering a composition comprising the multifunctional molecule of any one of claims 1-81.

90. The method of claim 89, wherein said cancer is selected from the group consisting of renal clear cell carcinoma (RCC), lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, gastric adenocarcinoma, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer (TNBC), head and neckDocket No. BREACH-001 / WOOl tumor, colorectal adenocarcinoma, melanoma, metastatic melanoma, gallbladder carcinoma, liver carcinoma, colon carcinoma, pancreatic carcinoma, bone sarcoma, thyroid adenocarcinoma, and renal cell carcinoma.

91. The method of treatment according to claim 89 or claim 90, wherein the patient experiences an increase in tumor growth inhibition of at least about 10%, 20%, 30% 40% 50% 60% 70% 80% 90%, 100%, 125%, 150%, 175%, 200% 225% 250%, 275%, 300%, 325% 350%, 375%, 400%, 425%, 450%, 475%, 500%, 525%, 550%, 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000%, as compared to a control or an untreated patient.

92. The method of treatment according to any one of claims 89-91, wherein the patient experiences a decrease in tumor growth inhibition of at least about 10%, 20%, 30% 40% 50% 60% 70% 80% 90%, 100%, 125%, 150%, 175%, 200% 225% 250%, 275%, 300%, 325% 350%, 375%, 400%, 425%, 450%, 475%, 50Q%, 525%, 550%, 575%, 600%, 625%, 650%, 675%, 700%, 725%, 750%, 775%, 800%, 825%, 850%, 875%, 900%, 925%, 950%, 975%, or 1000%, as compared to a control or an untreated patient.

93. A method of reducing a tumor comprising contacting the tumor with a composition comprising a multifunctional molecule of any one of claims 1-81.

94. A method of reducing a tumor in a subject in need thereof comprising administering to the subject a composition comprising a multifunctional molecule of any one of claims 1-81.

95. The method of any one of claims 87-94, wherein the subject is a human subject.

96. The multifunctional molecule of any one of claims 1-81, wherein the multifunctional molecule inhibits tumor growth in a mouse model.

97. The multifunctional molecule of claim 96, wherein the multifunctional molecule can reduce tumor growth by 60%.

98. The multifunctional molecule of any one of claims 96-97, wherein the multifunctional molecule comprises LIGHT.

99. The multifunctional molecule of any one of claims 96-98, wherein the multifunctional molecule comprises a tumor targeting moiety.

100. The multifunctional molecule of any one of claims 96-99, wherein the tumor targeting moiety targets PD-L1.

101. The multifunctional molecule of any one of claims 1-81, wherein the multifunctional molecule is cytotoxic to cancer cells.Docket No. BREACH-001 AVO01102. The multifunctional molecule of claim 101, wherein the multifunctional molecule further comprises a tumor-targeting moiety, optionally wherein the tumor targeting moiety targets DLL3, optionally wherein the tumor targeting moiety comprises an Fv or a VHH.

103. The multifunctional molecule of claim 101 or claim 102, wherein the multifunctional molecule further comprises an immune cell engager, optionally wherein the immune cell engager targets CD3.

104. The multifunctional molecule of any one of claims 101-103, wherein the multifunctional molecule further comprises a half-life extension moiety, optionally wherein the half-life extension moiety comprises Human Serum Albumin (HSA) binding protein such as a VHH.

105. The multifunctional molecule of any prior claim, wherein the trimeric ligand does not retain its ability to bind to its cognate receptors.

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