Intralesional injection of CpG ODNs complexed with glatiramer acetate mitigates systemic cytokine toxicities and synergistically advances checkpoint blockade efficacy.

PD-L1/PD-1 checkpoint inhibitors (CPIs) are mainstream agents for cancer immunotherapy, but the prognosis is unsatisfactory in solid tumor patients lacking preexisting T-cell reactivity. Adjunct therapy strategies including the intratumoral administration of immunostimulants aim to address this limitation. CpG oligodeoxynucleotides (ODNs), TLR9 agonists that can potentiate adaptive immunity, have been widely investigated to tackle PD-L1/PD-1 resistance, but clinical success has been hindered by inconsistent efficacy and immune-related toxicities caused by systemic exposure. Here, we utilized glatiramer acetate (GA), the FDA-approved, lysine-rich polypeptides to complex CpG into polycationic nanoparticles (R4B) and investigated the safety and antitumor efficacy of CpG ODNs in the murine CT26 colorectal carcinoma model. In a maximum tolerated dose study, repetitive R4B treatment displayed comparable antitumor efficacy to CpG alone treatment within a dose range from 15 µg to 150 µg while significantly attenuating systemic proinflammatory cytokine IL-6 release. A pharmacokinetic and biodistribution analysis confirmed that R4B localized and gradually released CpG around the lesions within 96 h while 'naked' CpG quickly diffused from the injection site. Genome-wide transcriptome analysis validated that R4B treatment activated prominent TLR9-driven immune system responses in both lesions and spleens. In a CT26 multiple tumor model, intratumoral administration of R4B generated systemic immune efficacy, evidenced by an abscopal effect on untreated tumors. Notably, R4B treatment accomplished these effects with mitigated systemic proinflammatory cytokines when compared with CpG alone. We further discovered that combining R4B with anti-PD-1 treatment led to the most pronounced effects on tumor growth and longest benefits to survival time. Our investigation into possible mechanisms underlying this phenomenon included increased recruitment of cytotoxic CD8 T cells and natural killer (NK) cells to the tumor microenvironments and the reversal of PD-L1/PD-1 axis inhibition. In summary, these results warrant further investigation for safely improving clinical responses in CPI-resistant solid tumor patients with localized CpG ODN therapy.