Monoclonal antibodies that target the inhibitory immune checkpoint axis consisting of programmed cell death protein 1 (PD-1) and its ligand, PD-L1, have changed the immune-oncology field. We identified K2, an anti-human PD-L1 single-domain antibody fragment, that can enhance T cell activation and tumor cell killing. In this study, the potential of different K2 formats as immune checkpoint blocking medicines was evaluated using a gene-based delivery approach. We showed that 2K2 and 3K2, a bivalent and trivalent K2 format generated using a 12 GS (glycine-serine) linker, were 313-and 135-fold more potent in enhancing T cell receptor (TCR) signaling in PD-1 POS cells than was monovalent K2. We further showed that bivalent constructs generated using a 30 GS linker or disulfide bond were 169and 35-fold less potent in enhancing TCR signaling than was 2K2. 2K2 enhanced tumor cell killing in a 3D melanoma model, albeit to a lesser extent than avelumab. Therefore, an immunoglobulin (Ig)G1 antibody-like fusion protein was generated, referred to as K2-Fc. K2-Fc was significantly better than avelumab in enhancing tumor cell killing in the 3D melanoma model. Overall, this study describes K2-based immune checkpoint medicines, and it highlights the benefit of an IgG1 Fc fusion to K2 that gains bivalency, effector functions, and efficacy.

Targeted radionuclide therapy (TRT) using probes labeled with Lutetium-177 represents a new and growing type of cancer therapy. We studied immunological changes in response to TRT with Lutetium-177 labeled anti-human CD20 camelid single domain antibodies (sdAbs) in a B16-melanoma model transfected to express human CD20, the target antigen, and ovalbumin, a surrogate tumor antigen. High-dose TRT induced melanoma cell death, calreticulin exposure and ATP-release in vitro. Melanoma-bearing mice received fractionated low and high-dose TRT via tumor targeting anti-human CD20 sdAbs, as opposed to control sdAbs. Tumor growth was delayed with both doses. Low and high-dose TRT increased interleukin-10 serum levels. Low-dose TRT also decreased CCL5 serum levels. At the tumor, high-dose TRT induced a type I interferon gene signature, while low-dose TRT induced a pro-inflammatory gene signature. Low and high-dose TRT increased the percentage of PD-L1pos and PD-L2pos myeloid cells in tumors with a marked increase in alternatively activated macrophages after high-dose TRT. The percentage of tumor-infiltrating T-cells was not changed, yet a modest increase in ovalbumin-specific CD8pos T-cells was observed after low-dose TRT. Contradictory, low and high-dose TRT decreased CD4pos T helper 1 (Th1)-cells in addition to double negative T-cells. In conclusion, these data suggest that low and high-dose TRT induce distinct immunological changes, which might serve as an anchoring point for combination therapy.

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Targeted radionuclide therapy (TRT) using targeting moieties labeled with a-particle-emitting radionuclides (a-TRT) is an intensely investigated treatment approach as the short range of a-particles allows effective treatment of local lesions and micrometastases. However, profound assessment of the immunomodulatory effect of a-TRT is lacking in literature. Methods: Using flow cytometry of tumors, splenocyte restimulation, and multiplex analysis of blood serum, we studied immunologic responses ensuing from TRT with an antihuman CD20 single-domain antibody radiolabeled with 225 Ac in a human CD20 and ovalbumin expressing B16-melanoma model. Results: Tumor growth was delayed with a-TRT and increased blood levels of various cytokines such as interferon-g, C-C motif chemokine ligand 5, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1. Peripheral antitumoral T-cell responses were detected on a-TRT. At the tumor site, a-TRT modulated the cold tumor microenvironment (TME) to a more hospitable and hot habitat for antitumoral immune cells, characterized by a decrease in protumoral alternatively activated macrophages and an increase in antitumoral macrophages and dendritic cells. We also showed that a-TRT increased the percentage of programmed death-ligand 1 (PD-L1)positive (PD-L1 pos ) immune cells in the TME. To circumvent this immunosuppressive countermeasure we applied immune checkpoint blockade of the programmed cell death protein 1-PD-L1 axis. Combination of a-TRT with PD-L1 blockade potentiated the therapeutic effect, however, the combination aggravated adverse events. A longterm toxicity study revealed severe kidney damage ensuing from a-TRT. Conclusion: These data suggest that a-TRT alters the TME and induces systemic antitumoral immune responses, which explains why immune checkpoint blockade enhances the therapeutic effect of a-TRT. However, further optimization is warranted to avoid adverse events.

Lung cancer thrives in a complex multicellular tumor microenvironment (TME) that impacts tumor growth, metastasis, response, and resistance to therapy. While orthotopic murine lung cancer models can partly recapitulate this complexity, they do not resonate with high‐throughput immunotherapeutic drug screening assays. To address the current need for relevant and easy‐to‐use lung tumor models, a protocol is established to generate and evaluate fully histocompatible murine and human lung tumor spheroids, generated by coculturing lung fibroblasts with tumor cells in ultralow adherence 96‐well plates. A spheroid generation protocol with the murine KrasG12Dp53−/− (KP) and Lewis Lung Carcinoma (LLC) cell lines is delivered next to the human lung H1650 adenocarcinoma line. In addition, their application potential to study tumor‐stroma organization, T‐cell motility, and infiltration as well as distinct macrophage subsets’ behavior using confocal microscopy is described. Finally, a 3D target‐specific T‐cell killing assay that allows spatiotemporal assessment of different tumor to T‐cell ratios and immune checkpoint blockade regimens using flow cytometry and live cell imaging is described. This 3D lung tumor spheroid platform can serve as a blueprint for other solid cancer types to comply with the need for straightforward murine and human oncoimmunology assays.