Agonistic Bivalent Human scFvs-Fcγ Fusion Antibodies to OX40 Ectodomain Enhance T Cell Activities against Cancer.

(1) Background: Understanding how advanced cancers evade host innate and adaptive immune opponents has led to cancer immunotherapy. Among several immunotherapeutic strategies, the reversal of immunosuppression mediated by regulatory T cells in the tumor microenvironment (TME) using blockers of immune-checkpoint signaling in effector T cells is the most successful treatment measure. Furthermore, agonists of T cell costimulatory molecules (CD40, 4-1BB, OX40) play an additional anti-cancer role to that of checkpoint blocking in combined therapy and serve also as adjuvant/neoadjuvant/induction therapy to conventional cancer treatments, such as tumor resection and radio- and chemo- therapies. (2) Methods and Results: In this study, novel agonistic antibodies to the OX40/CD134 ectodomain (EcOX40), i.e., fully human bivalent single-chain variable fragments (HuscFvs) linked to IgG Fc (bivalent HuscFv-Fcγ fusion antibodies) were generated by using phage-display technology and genetic engineering. The HuscFvs in the fusion antibodies bound to the cysteine-rich domain-2 of the EcOX40, which is known to be involved in OX40-OX40L signaling for NF-κB activation in T cells. The fusion antibodies caused proliferation, and increased the survival and cytokine production of CD3-CD28-activated human T cells. They showed enhancement trends for other effector T cell activities like granzyme B production and lysis of ovarian cancer cells when added to the activated T cells. (3) Conclusions: The novel OX40 agonistic fusion antibodies should be further tested step-by-step toward their safe use as an adjunctive non-immunogenic cancer immunotherapeutic agent.