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Natural killer (NK) cell therapy represents an attractive immunotherapy approach against recurrent epithelial ovarian cancer (EOC), as EOC is sensitive to NK cell-mediated cytotoxicity. However, NK cell antitumor activity is dampened by suppressive factors in EOC patient ascites. Here, we integrated functional assays, soluble factor analysis, high-dimensional flow cytometry cellular component data and clinical parameters of advanced EOC patients to study the mechanisms of ascites-induced inhibition of NK cells. Using a suppression assay, we found that ascites from EOC patients strongly inhibits peripheral blood-derived NK cells and CD34+ progenitor-derived NK cells, albeit the latter were more resistant. Interestingly, we found that higher ascites-induced NK cell inhibition correlated with reduced progression-free and overall survival in EOC patients. Furthermore, we identified transforming growth factor (TGF)-β1 to correlate with ascites-induced NK cell dysfunction and reduced patient survival. In functional assays, we showed that proliferation and anti-tumor reactivity of CD34+ progenitor-derived NK cells are significantly affected by TGF-β1 exposure. Moreover, inhibition of TGF-β1 signaling with galunisertib partly restored NK cell functionality in some donors. For the cellular components, we showed that the secretome is associated with a different composition of CD45+ cells between ascites of EOC and benign reference samples with higher proportions of macrophages in the EOC patient samples. Furthermore, we revealed that higher TGF-β1 levels are associated with the presence of M2-like macrophages, B cell populations and T-regulatory cells in EOC patient ascites. These findings reveal that targeting TGF-β1 signaling could increase NK cell immune responses in high-grade EOC patients.

Background While immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics. Results To introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with 111 In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy. Conclusions A set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets. Graphical Abstract

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas which secrete antibodies in the preferred format, species and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab fragments, isotype switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemo-enzymatic modification. We believe this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.

Many immunotherapies focus on (re)invigorating CD8+ T cell anti-cancer responses and different nuclear imaging techniques have been developed to measure CD8+ T cell distributions. In vivo labeling approaches using radiotracers primarily show CD8+ T cell distributions, while ex vivo labeled CD8+ T cells can show CD8+ T cell migration patterns, homing, and tumor infiltration. Currently, a comprehensive head-to-head comparison of in vivo and ex-vivo cell labeling with respect to their tumor and normal tissue targeting properties and correlation to the presence of CD8+ T cells is lacking, yet essential for correct interpretation of clinical CD8+ imaging applications. Therefore, we performed a head-to-head comparison of three different CD8+ T cell imaging approaches: 1) 89Zr-labeled DFO-conjugated Fc-silent anti-CD8 antibody ([89Zr]Zr-anti-CD8-IgG2asilent), 2) ex vivo 89Zr-oxine labeled ovalbumin-specific CD8+ T cells ([89Zr]Zr-OT-I cells), and 3) 18F-labeled IL2 ([18F]AlF-RESCA-IL2). B16F10/OVA tumor-bearing C57BL/6 mice (n=10/group) received intravenously one of the three radiopharmaceuticals. PET/CT images were acquired starting 72 h ([89Zr]Zr-anti-CD8-IgG2asilent), 24 and 48 h ([89Zr]Zr-OT-I cells), and 10 min ([18F]AlF-RESCA-IL2) post injection. Subsequently, ex vivo biodistribution analysis of the radiopharmaceuticals was performed followed by flow cytometric analysis to evaluate the number of intratumoral CD8+ T cells. Additionally, the intratumoral radiolabel distributions was assessed by autoradiography and immunohistochemistry (IHC) on tumor slices. [89Zr]Zr-anti-CD8-IgG2asilent, [89Zr]Zr-OT-I cells, and [18F]AlF-RESCA-IL2 showed uptake in CD8-rich tissues, with preferential targeting to the spleen. Biodistribution analysis showed tumor uptake above blood level for all radiopharmaceuticals, except [18F]AlF-RESCA-IL2. For all three approaches, the uptake in the tumor-draining lymph node was significantly higher compared with the contralateral axial lymph node, suggesting that all approaches allow evaluation of immune responses involving CD8+ T cells. Tumor uptake of [89Zr]Zr-anti-CD8-IgG2asilent (R2=0.65, p<0.01) and [89Zr]Zr-OT-I cells (R2=0.74, p<0.01) correlated to the number of intratumoral CD8+ T cells (flow cytometry). The intratumoral distribution pattern of the radiosignal was different for ex vivo and in vivo radiolabeling techniques. The short half-life of 18F precluded autoradiography assessment of [18F]AlF-RESCA-IL2. We show that [89Zr]Zr-anti-CD8-IgG2asilent and [89Zr]Zr-OT-I cells PET/CT imaging can be used to evaluate intratumoral CD8+ T cells, even though their normal tissues and intratumoral distribution patterns are significantly different. Based on their characteristics, [89Zr]Zr-anti-CD8-IgG2asilent might be most useful to immunophenotyping the TME, while the ex vivo cell labeling approach visualizes CD8+ T cell migrations patterns and the permissiveness of tumors for invasion, whereas [18F]AlF-RESCA-IL2 allows for rapid recurrent imaging and might prove useful for tracking rapid changes in CD8+ T cell distributions. In conclusion, our head-to-head comparison of the three prototype CD8+ T cell labeling approaches provides new insights which can aid in correct interpretation of clinical CD8 imaging and may guide in the selection of the optimal imaging approach for the research question of interest.