Engineered soluble, trimerized 4-1BBL variants as potent immunomodulatory agents

Targeting co-stimulatory receptors promotes the activation and effector functions of anti-tumor lymphocytes. 4-1BB (CD137/TNFSF9), a member of the tumor necrosis factor receptor superfamily (TNFR-SF), is a potent co-stimulatory receptor that plays a prominent role in augmenting effector functions of CD8⁺ T cells, but also CD4⁺ T cells and NK cells. Agonistic antibodies against 4-1BB have entered clinical trials and shown signs of therapeutic efficacy. Here, we have used a T cell reporter system to evaluate various formats of 4-1BBL regarding their capacity to functionally engage its receptor. We found that a secreted 4-1BBL ectodomain harboring a trimerization domain derived from human collagen (s4-1BBL-Tri_XVIII) is a strong inducer of 4-1BB co-stimulation. Similar to the 4-1BB agonistic antibody urelumab, s4-1BBL-Tri_XVIII is very potent in inducing CD8⁺ and CD4⁺ T cell proliferation. We provide first evidence that s4-1BBL-Tri_XVIII can be used as an effective immunomodulatory payload in therapeutic viral vectors. Oncolytic measles viruses encoding s4-1BBL-Tri_XVIII significantly reduced tumor burden in a CD34⁺ humanized mouse model, whereas measles viruses lacking s4-1BBL-Tri_XVIII were not effective. Natural soluble 4-1BB ligand harboring a trimerization domain might have utility in tumor therapy especially when delivered to tumor tissue as systemic administration might induce liver toxicity.