A recombinant, trispecific monovalent antibody-based molecule targeting the human programmed death-ligand 1 (PD-L1), 4-1BB (CD137; tumor necrosis factor receptor superfamily member 9; TNFRSF9) and human serum albumin (HSA), with potential checkpoint inhibitory, immunostimulating and antineoplastic activities. PD-L1/4-1BB/HSA trispecific fusion protein NM21-1480 consists of three monovalent antibody Fvs specific for PD-L1, HSA and 4-1BB fused in a single chain. Upon administration, PD-L1/4-1BB/HSA trispecific fusion protein NM21-1480 simultaneously targets and binds to a membrane-distal epitope of 4-1BB, which is expressed on a variety of leukocyte subsets including activated T-lymphocytes, and PD-L1 expressed on tumor cells. The simultaneous binding to PD-L1 enables clustering of 4-1BB and thereby allows for conditional stimulation of 4-1BB signaling in the tumor microenvironment (TME) only upon binding to PD-L1 on tumor cells. 4-1BB activation results in T-cell stimulation and enhances T-lymphocyte-mediated anti-tumor activity. At the same time, NM21-1480 prevents PD-L1 from binding to and activating its receptor, programmed cell death 1 (PD-1; PDCD1; CD279; programmed death-1). This abrogates T-cell inhibition, activates antigen-specific T-lymphocytes and enhances cytotoxic T-cell-mediated tumor cell lysis, which may lead to a reduction in tumor growth. PD-L1 binding to PD-1 on activated T-cells inhibits the expansion and survival of CD8-positive T-cells, suppresses the immune system and results in immune evasion. 4-1BB, a surface glycoprotein of the tumor necrosis factor receptor superfamily, is an inducible costimulatory receptor that plays a key role in T-cell proliferation, survival and cytolytic activity. HSA fusion prolongs the half-life of NM21-1480. The conditional activation of 4-1BB signaling prevents systemic T-cell activation and lowers toxicity.