Targeted Activation of Toll-Like Receptors: Conjugation of a Toll-Like Receptor 7 Agonist to a Monoclonal Antibody Maintains Antigen Binding and Specificity

Therapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of antitumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to nonspecific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an antitumor mAb (the anti-hCD20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods, and yield, molecular substitution ratio, retention of TLR7 activity, and specificity of antigen binding were compared. Both conjugation methods produced rituximab–UC-1V150 conjugates with UC-1V150: rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional cross-linker was first reacted with rituximab and second to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65–78%. Rituximab–UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28–53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab–UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells while no nonspecific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible.