Tumor necrosis factor superfamily and TNF-like ligand muteins and methods of preparing

A trimer comprising monomers of a soluble polypeptide, wherein said soluble polypeptide comprises a tumor necrosis factor superfamily (TNFSF) ligand or TNF-like ligand, or an extracellular domain thereof, wherein at least a first said soluble polypeptide of the trimer comprises an amino acid substitution or insertion that introduces a surface-exposed, exterior-facing cysteine residue at a position in the first soluble polypeptide that is located at a natural interface between monomers of the trimer, wherein an alpha carbon (Cα) of the cysteine residue of the first soluble polypeptide is within 9 Å of a Cα of a cysteine residue of a second said soluble polypeptide of the trimer that is also located at the natural interface between the monomers of the trimer, wherein the cysteine residue of the first soluble polypeptide is disulfide bonded to the cysteine residue of the second soluble polypeptide, and wherein said TNFSF or TNF-like ligand is tumor necrosis factor-α (TNF-α), lymphotoxin-α (LT-α), lymphotoxin-β (LT-β), cluster of differentiation 40 ligand (CD40L), cluster of differentiation 70 (CD70), receptor activator of nuclear factor kappa beta ligand (RANKL), tumor necrosis family receptor superfamily member 4 ligand (OX40L), cluster of differentiation 95 ligand (FasL), tumor necrosis family receptor superfamily member 9 (4-1BB ligand), TNF-related apoptosis-inducing ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK), a proliferation-inducing ligand (APRIL), B lymphocyte stimulator (BLys), tumor necrosis family receptor superfamily member 14 (LIGHT), TNF-like ligand 1 (TL1), glucocorticoid-induced TNF receptor ligand (GITRL), ectodysplasin A1 (EDA-A1), ectodysplasin A2 (EDA-A2), or adiponectin.