Radiolabeled single-domain antibodies as a means to evoke and monitor immunological responses in malignant lesions

Cancer is a complex and heterogeneous disease in which cells divide uncontrollably and remains a significant challenge to diagnose and treat. Current treatment approaches lack specificity, unable to distinguish between healthy and diseased cells, resulting in either debilitating side-effects or incomplete ablation of malignant cells. Hence, novel anti-cancer compounds incorporate targeting, which limits side-effects due to off-target binding to healthy cells. In the past few years, single-domain antibodies (sdAbs), the smallest antigen binding fragment of heavy chain only monoclonal antibodies (HCAbs), have gathered renown as a targeting vector. sdAbs have excellent properties such as fast pharmacokinetics, allow to be conjugated with various radionuclides, bind to their cognate antigens and thanks to their small size, rapidly clear and diffuse in tissue upon administration. Recently, the first set of clinical trials involving radiolabeled sdAbs were successfully completed, providing proof of concept (PoC) of their use in a real-life scenario, be it for imaging or therapy. In this thesis, radiolabeled sdAbs were assessed for their ability to (i) document immunological changes in malignancy using nuclear medicine imaging and (ii) achieve immune-mediated therapeutic anti-cancer effects using targeted radionuclide therapy (TRT).
Studies were performed in immunocompetent, murine animal models, engrafted with modified melanoma cells to express the model antigen ovalbumin, a well-characterized Gallus gallus domesticus antigen. (i) Immune imaging was performed using technetium-99m (99mTc) radiolabeled anti-murine programmed death-ligand 1 (PD-L1) sdAbs upon vaccination with an mRNA vaccine, called Galsomes. (ii) In TRT-experiments, the melanoma cells also expressed the target antigen human CD20 (huCD20), to allow TRT with either β--emitter Lutetium-177 (177Lu) or α-emitter, Actinium-225 (225Ac) radiolabeled anti-huCD20 sdAbs. Additionally, various read-outs were performed on blood, tumor tissue and healthy organs to assess immune parameters.
(i) Immune imaging documented the dynamic expression of PD-L1 in a mouse melanoma model after Galsome vaccination. We showed the ability to generate serial high-resolution images allowing quantification of PD-L1 expression in peripheral organs and tumors. Ex vivo analysis using flow cytometry confirmed the results of the quantified images and showed that both non-immune and myeloid cells contributed to PD-L1 expression in the organs that were studied. (ii) TRT-experiments showed a delay in tumor growth in both α- and β--TRT which translated into a prolonged time to reach humane endpoint. Intriguingly, β--TRT induced protumoral, suppressive immune responses as opposed to α-TRT, which exhibited an increase in anti-tumoral immune activity. More specifically, β--TRT was characterized by increased interleukin (IL)-10 serum levels, increased the percentage of PD-L1pos and PD-L2pos myeloid cells in tumors with a marked increase in alternatively activated macrophages and decreased CD4pos T helper 1 (TH1)-cells. α-TRT was characterized by changes in various blood cytokine levels, a decrease in pro-tumoral alternatively activated macrophages and an increase in antitumoral macrophages and dendritic cells (DCs). We also showed that α-TRT increased the percentage of PD-L1pos immune cells in the tumor, providing a rationale for performing cotreatment with checkpoint blockade of the PD-1|PD-L1 axis. Combination of α-TRT with PDL1 blockade potentiated the therapeutic effect, however aggravated adverse events and a longterm toxicity study revealed severe kidney damage ensuing α-TRT.
To conclude, in this dissertation we documented immunological changes using 99mTc-labeled anti-PD-L1 sdAbs, marked by an increase in PD-L1 expression of lung and liver tissue upon administration of Galsomes, however the results obtained from tumor tissue remain ambiguous. As for immunological changes upon TRT, we documented a difference in response to α- and β--TRT. Whereas alterations in the TME upon α-TRT were more in favor of anti-tumoral immune responses, β--TRT induced an increase in immunosuppressive populations. Future perspectives for this thesis would be to optimize sdAb-based PD-L1 imaging as well as delve further into the immunological field when TRT is combined with promising immune and non-immune therapies and include upcomming immune checkpoint to these read-outs.