S1 Dataset -

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.

咪喹莫特（Imiquimod）是一种Toll样受体7（Toll-like receptor 7, TLR7）激动剂，常规用于基底细胞癌与零期黑色素瘤的局部给药治疗。同理，TLR激动剂卡介苗（Bacillus Calmette-Guérin, BCG）可用于膀胱癌的局部治疗，临床研究证实瘤内注射Toll样受体9（Toll-like receptor 9, TLR9）激动剂亦具有治疗效果。然而，若经全身给药，内体TLR激动剂会因广泛的免疫激活引发不良反应。因此，亟需开发将TLR激动剂靶向递送至肿瘤组织的策略，以推动内体TLR激动剂在肿瘤免疫治疗中的广泛应用。靶向递送TLR激动剂的策略之一，是将其与肿瘤抗原特异性治疗性抗体进行偶联。此类抗体-TLR激动剂偶联物可通过诱导局部TLR介导的先天免疫激活发挥协同作用，与治疗性抗体诱导的抗肿瘤免疫机制形成互补。本研究探索了将TLR9激动剂与免疫球蛋白G（immunoglobulin G, IgG）偶联的多种不同策略。我们对比了随机偶联与位点特异性偶联两种方式，评估了使用不同交联剂将免疫刺激性CpG寡脱氧核苷酸（CpG oligodesoxyribonucleotides, ODN）与HER2特异性治疗性抗体曲妥珠单抗（Trastuzumab）进行生化偶联的效果。我们在体外对所制备的曲妥珠单抗-CpG ODN偶联物的理化特性与生物学活性进行了表征，结果证实，CpG ODN的位点特异性偶联对于维持曲妥珠单抗的抗原结合能力至关重要。进一步研究表明，位点特异性偶联物可在携带工程化人源HER2转基因肿瘤细胞的伪转移小鼠模型中，有效促进体内抗肿瘤免疫应答。在该体内模型中，以位点特异性偶联物形式共递送曲妥珠单抗与CpG ODN，在促进T细胞活化与增殖方面，效果优于未偶联的曲妥珠单抗、CpG ODN单独给药或随机偶联物的联合注射。综上，本研究表明，将CpG ODN位点特异性偶联至靶向肿瘤标志物的治疗性抗体，是一种可行且更可靠的偶联物制备方法，该方法可保留并结合佐剂与抗体的双重功能特性。