Dendritic cells (DC) are crucial for the priming of T cells and thereby influence adaptive immune responses. Hence, they also represent important players in shaping anti-tumour immune responses. Cancer immunotherapy has been driven over many years by the aim to harness the T-cell stimulatory activity of these crucial antigen-presenting cells (APC). Efficient antigen delivery alone is not sufficient for full engagement of the T-cell stimulatory activity of DC and the inclusion of adjuvants triggering appropriate DC activation is essential to ensure effective anti-tumour immunity induction. While the direct engagement of DC function is a powerful tool for tumour immunotherapy, many therapeutic antibodies, such as antibodies directed against tumour-associated antigens (TAA) and immune checkpoint inhibitors (ICI) have been shown to engage DC function indirectly. The induction of anti-tumour immune responses by TAA-targeting and immune checkpoint inhibitory antibodies is thought to be integral to their therapeutic efficacy. Here, we provide an overview of the immunotherapeutic antibodies in the context of cancer immunotherapy, that has been demonstrated to directly or indirectly engage DC and discuss the current understanding of the functional mechanisms underlying anti-tumour immunity induction by these antibody therapies. In the future, the combination of therapeutic strategies that engage DC function directly and/or indirectly with strategies that allow tumour infiltrating immune effector cells to exert their anti-tumour activity in the tumour microenvironment (TME) may be key for the successful treatment of cancer patients currently not responding to immunotherapeutic antibody treatment.

Imiquimod, a Toll-like receptor 7 (TLR7) agonist is routinely used for topical administration in basal cell carcinoma and stage zero melanoma. Similarly, the TLR agonist Bacillus Calmette-Guérin is used for the local treatment of bladder cancer and clinical trials showed treatment efficacy of intratumoral injections with TLR9 agonists. However, when administered systemically, endosomal TLR agonists cause adverse responses due to broad immune activation. Hence, strategies for targeted delivery of TLR agonists to the tumor tissue are needed to enable the widespread use of endosomal TLR agonists in the context of tumor immunotherapy. One strategy for targeted delivery of TLR agonist is their conjugation to tumor antigen-specific therapeutic antibodies. Such antibody-TLR agonist conjugates act synergistically by inducing local TLR-mediated innate immune activation which complements the anti-tumor immune mechanisms induced by the therapeutic antibody. In this study, we explored different conjugation strategies for TLR9 agonists to immunoglobulin G (IgG). We evaluated biochemical conjugation of immunostimulatory CpG oligodesoxyribonucleotides (ODN) to the HER2-specific therapeutic antibody Trastuzumab with different cross-linkers comparing stochastic with site-specific conjugation. The physiochemical make-up and biological activities of the generated Trastuzumab-ODN conjugates were characterized in vitro and demonstrated that site-specific conjugation of CpG ODN is crucial for maintaining the antigen-binding capabilities of Trastuzumab. Furthermore, site-specific conjugate was effective in promoting anti-tumor immune responses in vivo in a pseudo-metastasis mouse model with engineered human HER2-transgenic tumor cells. In this in vivo model, co-delivery of Trastuzumab and CpG ODN in form of site-specific conjugates was superior to co-injection of unconjugated Trastuzumab, CpG ODN or stochastic conjugate in promoting T cell activation and expansion. Thereby, this study highlights that site-specific conjugation of CpG ODN to therapeutic antibodies targeting tumor markers is a feasible and more reliable approach for generation of conjugates which retain and combine the functional properties of the adjuvant and the antibody.