Materials and methods of using engineered ligands

#Described herein are compositions and kits that comprise an engineered TL1A ligand that displays high stability, minimal binding to decoy receptor DcR3 while retaining functional activity via binding to its cell surface receptor, DR3, and the ability to activate T cells in vitro and in vivo. Methods of making an engineered TL1A ligand and methods of treating a disease or disorder in a subject by administering an engineered TL1A ligand are also provided.

#This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name of “14620-570-999_SUB_SEQ_LISTING.txt” and a creation date of Mar. 9, 2023 and having a size of 324,576 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

#The tumor necrosis factor family member TNF-like factor 1A (TL1A) co-stimulatory receptor (also known as TNF superfamily member 15 (TNFSF15)) is expressed on a variety of cell types as a type II single-pass transmembrane protein. It can be cleaved from the cell surface, and soluble or membrane-bound TL1A binds to the co-stimulatory death receptor 3 (DR3). TL1A can also be bound up by the soluble decoy receptor DcR3 (TNFRSF6B), which mimics the structure of cell surface receptors but lacks a transmembrane or cytoplasmic region. DcR3 is structurally homologous to DR3 but shares only approximately 20% sequence identity with DR3. DcR3 is up-regulated in a variety of tumors and is associated with metastasis (Hsieh and Lin, 2017; Lin and Hsieh, 2011; Wei et al., 2019; Zhang et al., 2017). DcR3 decoy receptors can serve as a sink to prevent TNF ligands from activating T cells, and soluble decoy receptors can be up-regulated by tumors. Specifically, the decoy receptor DcR3 can bind to TL1A with higher affinity than its cell surface receptor, DR3, and can prevent TL1A-based T cell costimulation (Hsieh and Lin, 2017; Lin and Hsieh, 2011).

#FIGS. 1A-1E depict the design of constructs. (A) Schematic diagram of TL1A, including the transmembrane region (TM) from residues 35 to 56 and the tumor necrosis factor homology domain (TNFHD) from residues 80 to 250. Residues N133 and N229 are glycosylation sites and residues C162 and C202 form a disulfide bond. (B) Ribbon diagram depiction of a TL1A monomer. (C) TL1A ligands were expressed having a C-terminal His-tag as a non-covalent trimer (TL1A) or as a single-chain trimer (scTL1A) (left and right panels, respectively). (D) TL1A was expressed as a fusion to the C-terminus of a human IgG1 Fc (Fc-TL1A), an Fc-scTL1A, or by co-expression of Fc-TL1A with His-TL1A (Fc-His-TL1A) (left, middle, and right panels, respectively). (E) TL1A was expressed to a HSA monomer (HSA-TL1A) or HSA-scTL1A (left and right panels, respectively).

#FIGS. 2A-2F depict the analysis of the oligomeric state and function of TL1A constructs. (A) Preparative gel-filtration analysis of indicated TL1A constructs. Recombinant TNFα (His-scTNFα) was used as a control to show the population of trimers and oligomers. (B) Analytical SEC analysis of indicated TL1A constructs after preparative gel-filtration purification to isolate desired oligomeric species. The purified species were used in functional assays. (C)-(D) ELISA analysis of the ability of TL1A constructs to bind immobilized DR3 (C) or DcR3 (D). Molar concentrations of TL1A for each construct were normalized to the concentration of TL1A trimers in each molecule. (E)-(F) Pan T cells from healthy donors were incubated with plate-bound anti-CD3 antibody (0.01 μg/mL). Indicated TL1A ligands were added at 0, 0.03, 0.1, 0.3, 1, 3, 10, 30 or 100 nM (trimer molarity) to α-CD3-activated T cells (bars from left to right). Levels of IFNγ (E) and TNFα (F) produced by the activated T cells are shown.

#FIG. 3 depicts that Fc-TL1A ligands activate T cells in vivo. Mice were treated with anti-CD3 antibody and Fc-TL1A or Fc-scTL1A and serum concentrations of IFNγ were measured. IFNγ levels are shown in pg/mL and constructs are indicated on the graph.

#The tumor necrosis factor family member TNF-like factor 1A (TL1A), also known as TNF-like protein 1A (TNFSF15), is a homo-trimeric TNF superfamily ligand. TL1A is expressed on a variety of cell types as a type II single-pass transmembrane protein. It can be cleaved from the cell surface to enter circulation as a soluble ligand and it binds to the T cell co-stimulatory death receptor 3 (DR3). TL1A can also be bound up by the soluble decoy receptor DcR3 (TNFRSF6B), which is structurally homologous to DR3 but shares only approximately 20% sequence identity with DR3.

#The present disclosure is based, at least in part, on the recognition of therapeutic strategy that promotes specific TL1A ligand binding to DR3 and not DcR3. Its potential as a co-stimulatory receptor to overcome T cell exhaustion in solid tumors makes DR3 an attractive immune checkpoint target.

#In some embodiments, engineered TL1A ligands for use with the methods described herein are capable of binding the TNFRSF member death receptor 3 (DR3). In some embodiments, engineered TL1A ligands disclosed herein are capable of binding DR3 with a 3:3 stoichiometry of interaction with DR3 receptors. In some embodiments, the engineered TL1A ligands bind disclosed herein bind specifically to DR3. In some embodiments, the engineered TL1A ligands disclosed herein bind to DR3 with affinity similar to that of wild-type TL1A. In some embodiments, the engineered TL1A ligands bind to DR3 with higher affinity than that of wild-type TL1A, e.g., by at least about 10% higher affinity, at least about 20% higher affinity, at least about 30% higher affinity, at least about 40% higher affinity, at least about 50% higher affinity, at least about 60% higher affinity, at least about 70% higher affinity, at least about 80% higher affinity, at least about 90% higher affinity, at least 100% higher affinity, at least 200% higher affinity, at least 400% higher affinity or more.