Eccentric: Enhancing the function of cancer countering effector cells using nanobodies targeting regulatory immune checkpoints

The immune system protects against infections and cells that acquire malignant traits through genetic errors. However, sometimes these malignant cells accumulate features that allow them to hide from the immune system, for instance overexpression of regulatory immune checkpoints (ICPs), which serve as a molecular cloak. The most renowned ICP axis contains the programmed death protein-1 (PD-1) and its ligand PD-L1. Once the immune system loses control over cancer cells, intervention with monoclonal antibodies (mAbs) targeting the PD-1:PD-L1-axis can bring the immune system back on track and stimulate immune cell-mediated tumor radication. ICP therapy revolutionized the field of oncology and became a mainstay therapy option for many malignancies. Despite promising results in many patients, the majority of patients cannot benefit from it. Low response rates and high toxicity prompted exploration of novel administration routes and delivery platforms for ICP drugs as well as new antibody formats with superior properties. In this PhD, we contributed to this line of research and explored delivery strategies for, and the therapeutic potential of the human PD-L1 targeting nanobody (Nb) K2. Moreover, we evaluated different strategies such as protein engineering and combination approaches to improve K2-based ICP therapy.
Since K2 is rapidly cleared upon systemic administration, its therapeutic efficacy might be limited in vivo. Therefore, finding an approach to extend the presence of K2 is required. To provide a continuous K2 supply, we formulated it into a biodegradable peptide hydrogel that can serve as
slow-release depot. Subcutaneous injection of the hydrogel into mice with melanoma, significantly prolonged the overall presence of K2 at the injection site, in the blood and in distant tumors. Although, the hydrogel prolonged K2 delivery, the release was limited to 24 hours. Since genebased delivery using lentiviral vectors (LVs) promises permanent modification of cells, we explored whether LV delivery of K2 to tumor cells can restore antitumor immunity. Genetic modification with K2-encoding LVs enabled in situ K2 production that resulted in immune cell activation. To increase the efficiency of K2 and thereby reducing the LV-load necessary for ICP blockade, we increased the valency by coupling two or three K2 molecules together and evaluated different linkers. Increasing the valency significantly reduced the LV amounts required for ICP blockade. Equipping K2 with a human immunoglobulin (Ig)G1 Fc-tail enabled antibody-dependent cellular toxicity (ADCC) and thereby increased the efficiency of LV-mediated K2 delivery. Since we observed that LV-delivered K2-Fc effectively antagonizes PD-1 and induces ADCC, we explored whether these effects can be augmented by LV-mediated interleukin (IL)-15 delivery. We demonstrated that LV-delivered IL-15 induced natural killer (NK) cell expansion, immune cell activation and tumor cell killing. We observed that K2-Fc and IL-15 cooperated in tumor cell killing
and immune cell activation and revealed an interplay between CD4+ T cells and NK cells.
To conclude, this thesis describes hydrogel and gene-based methods for delivery of PD-L1 blocking Nbs, how engineering of the Nb-format impacts the therapy efficiency and how combination with cytokine therapy improves the efficacy of gene-based anti-PD-L1 therapy. These findings pave the road for the development of novel Nb-based ICP drugs and delivery strategies.