Scrutiny of chimeric antigen receptor activation by the extracellular domain: experience with single domain antibodies targeting multiple myeloma cells highlights the need for case-by-case optimization.

Introduction: Multiple myeloma (MM) remains incurable, despite the advent of chimeric antigen receptor (CAR)-T cell therapy. This unfulfilled potential can be attributed to two untackled issues: the lack of suitable CAR targets and formats. In relation to the former, the target should be highly expressed and reluctant to shedding; two characteristics that are attributed to the CS1-antigen. Furthermore, conventional CARs rely on scFvs for antigen recognition, yet this withholds disadvantages, mainly caused by the intrinsic instability of this format. VHHs have been proposed as valid scFv alternatives. We therefore intended to develop VHH-based CAR-T cells, targeting CS1, and to identify VHHs that induce optimal CAR-T cell activation together with the VHH parameters required to achieve this. Methods: CS1-specific VHHs were generated, identified and fully characterized, in vitro and in vivo. Next, they were incorporated into second-generation CARs that only differ in their antigen-binding moiety. Reporter T-cell lines were lentivirally transduced with the different VHH-CARs and CAR-T cell activation kinetics were evaluated side-by-side. Affinity, cell-binding capacity, epitope location, in vivo behavior, binding distance, and orientation of the CAR-T:MM cell interaction pair were investigated as predictive parameters for CAR-T cell activation. Results: Our data show that the VHHs affinity for its target antigen is relatively predictive for its in vivo tumor-tracing capacity, as tumor uptake generally decreased with decreasing affinity in an in vivo model of MM. This does not hold true for their CAR-T cell activation potential, as some intermediate affinity-binding VHHs proved surprisingly potent, while some higher affinity VHHs failed to induce equal levels of T-cell activation. This could not be attributed to cell-binding capacity, in vivo VHH behavior, epitope location, cell-to-cell distance or binding orientation. Hence, none of the investigated parameters proved to have significant predictive value for the extent of CAR-T cell activation. Conclusions: We gained insight into the predictive parameters of VHHs in the CAR-context using a VHH library against CS1, a highly relevant MM antigen. As none of the studied VHH parameters had predictive value, defining VHHs for optimal CAR-T cell activation remains bound to serendipity. These findings highlight the importance of screening multiple candidates.

Nanobody-mediated SPECT/CT imaging reveals the spatiotemporal expression of programmed death-ligand 1 in response to a CD8+ T cell and iNKT cell activating mRNA vaccine.

Rationale: Although promising responses are obtained in patients treated with immune checkpoint inhibitors targeting programmed death ligand 1 (PD-L1) and its receptor programmed death-1 (PD-1), only a fraction of patients benefits from this immunotherapy. Cancer vaccination may be an effective approach to improve the response to immune checkpoint inhibitors anti-PD-L1/PD-1 therapy. However, there is a lack of research on the dynamics of PD-L1 expression in response to cancer vaccination. Methods: We performed non-invasive whole-body imaging to visualize PD-L1 expression at different timepoints after vaccination of melanoma-bearing mice. Mice bearing ovalbumin (OVA) expressing B16 tumors were i.v. injected with the Galsome mRNA vaccine: OVA encoding mRNA lipoplexes co-encapsulating a low or a high dose of the atypical adjuvant α-galactosylceramide (αGC) to activate invariant natural killer T (iNKT) cells. Serial non-invasive whole-body immune imaging was performed using a technetium-99m (99mTc)-labeled anti-PD-L1 nanobody, single-photon emission computerized tomography (SPECT) and X-ray computed tomography (CT) images were quantified. Additionally, cellular expression of PD-L1 was evaluated with flow cytometry. Results: SPECT/CT-imaging showed a rapid and systemic upregulation of PD-L1 after vaccination. PD-L1 expression could not be correlated to the αGC-dose, although we observed a dose-dependent iNKT cell activation. Dynamics of PD-L1 expression were organ-dependent and most pronounced in lungs and liver, organs to which the vaccine was distributed. PD-L1 expression in lungs increased immediately after vaccination and gradually decreased over time, whereas in liver, vaccination-induced PD-L1 upregulation was short-lived. Flow cytometric analysis of these organs further showed myeloid cells as well as non-immune cells with elevated PD-L1 expression in response to vaccination. SPECT/CT imaging of the tumor demonstrated that the expression of PD-L1 remained stable over time and was overall not affected by vaccination although flow cytometric analysis at the cellular level demonstrated changes in PD-L1 expression in various immune cell populations following vaccination. Conclusion: Repeated non-invasive whole-body imaging using 99mTc-labeled anti-PD-L1 nanobodies allows to document the dynamic nature of PD-L1 expression upon vaccination. Galsome vaccination rapidly induced systemic upregulation of PD-L1 expression with the most pronounced upregulation in lungs and liver while flow cytometry analysis showed upregulation of PD-L1 in the tumor microenvironment. This study shows that imaging using nanobodies may be useful for monitoring vaccine-mediated PD-L1 modulation in patients and could provide a rationale for combination therapy. To the best of our knowledge, this is the first report that visualizes PD-L1 expression upon cancer vaccination.

In vitro modelling of local gene therapy with IL-15/IL-15Rα and a PD-L1 antagonist in melanoma reveals an interplay between NK cells and CD4+ T cells.

Blockade of the immune checkpoint axis consisting of programmed death-1 (PD-1) and its ligand PD-L1 alleviates the functional inhibition of tumor-infiltrating lymphoid cells yet weakly induces their expansion. Exogenous cytokines could further expand lymphoid cells and thus synergize with αPD-L1 therapy. However, systemic delivery of most cytokines causes severe toxicity due to unspecific expansion of immune cells in the periphery. Here, we modelled local delivery of cytokines and αPD-L1 therapeutics to immune cell-containing in vitro melanoma tumors. Three-dimensional tumor models consisting of 624-MEL cells were co-cultured with human peripheral blood lymphoid cells (PBLs) in presence of the cytokines IL-2, IL-7, IL-15, IL-21 and IFN-γ. To model local gene therapy, melanoma tumors were modified with lentiviral vectors encoding IL-15 fused to IL-15Rα (IL-15/IL-15Rα) and K2-Fc, a fusion of a human PD-L1 specific single domain antibody to immunoglobulin (Ig)G1 Fc. To evaluate the interplay between PBL fractions, NK cells, CD4+ T cells or CD8+ T cells were depleted. Tumor cell killing was followed up using real time imaging and immune cell expansion and activation was evaluated with flow cytometry. Among the tested cytokines, IL-15 was the most potent cytokine in stimulating tumor cell killing and expanding both natural killer (NK) cells and CD8+ T cells. Gene-based delivery of IL-15/IL-15Rα to tumor cells, shows expansion of NK cells, activation of NK cells, CD4+ and CD8+ T cells, and killing of tumor spheroids. Both NK cells and CD8+ T cells are necessary for tumor cell killing and CD4+ T-cell activation was reduced without NK cells. Co-delivery of K2-Fc improved tumor cell killing coinciding with increased activation of NK cells, which was independent of bystander T cells. CD4+ or CD8+ T cells were not affected by the co-delivery of K2-Fc even though NK-cell activation impacted CD4+ T-cell activation. This study demonstrates that gene-based delivery of IL-15/IL-15Rα to tumor cells effectively mediates anti-tumor activity and sensitizes the tumor microenvironment for therapy with αPD-L1 therapeutics mainly by impacting NK cells. These findings warrant further investigation of gene-based IL-15 and K2-Fc delivery in vivo.

Targeted α-Therapy Using 225Ac Radiolabeled Single-Domain Antibodies Induces Antigen-Specific Immune Responses and Instills Immunomodulation Both Systemically and at the Tumor Microenvironment.

Targeted radionuclide therapy (TRT) using targeting moieties labeled with α-particle-emitting radionuclides (α-TRT) is an intensely investigated treatment approach as the short range of α-particles allows effective treatment of local lesions and micrometastases. However, profound assessment of the immunomodulatory effect of α-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 225Ac in a human CD20 and ovalbumin expressing B16-melanoma model. Results: Tumor growth was delayed with α-TRT and increased blood levels of various cytokines such as interferon-γ, C-C motif chemokine ligand 5, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1. Peripheral antitumoral T-cell responses were detected on α-TRT. At the tumor site, α-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 α-TRT increased the percentage of programmed death-ligand 1 (PD-L1)-positive (PD-L1pos) 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 α-TRT with PD-L1 blockade potentiated the therapeutic effect, however, the combination aggravated adverse events. A long-term toxicity study revealed severe kidney damage ensuing from α-TRT. Conclusion: These data suggest that α-TRT alters the TME and induces systemic antitumoral immune responses, which explains why immune checkpoint blockade enhances the therapeutic effect of α-TRT. However, further optimization is warranted to avoid adverse events.

Junctional adhesion molecule-A is dispensable for myeloid cell recruitment and diversification in the tumor microenvironment.

Junctional adhesion molecule-A (JAM-A), expressed on the surface of myeloid cells, is required for extravasation at sites of inflammation and may also modulate myeloid cell activation. Infiltration of myeloid cells is a common feature of tumors that drives disease progression, but the function of JAM-A in this phenomenon and its impact on tumor-infiltrating myeloid cells is little understood. Here we show that systemic cancer-associated inflammation in mice enhanced JAM-A expression selectively on circulating monocytes in an IL1ß-dependent manner. Using myeloid-specific JAM-A-deficient mice, we found that JAM-A was dispensable for recruitment of monocytes and other myeloid cells to tumors, in contrast to its reported role in inflammation. Single-cell RNA sequencing revealed that loss of JAM-A did not influence the transcriptional reprogramming of myeloid cells in the tumor microenvironment. Overall, our results support the notion that cancer-associated inflammation can modulate the phenotype of circulating immune cells, and we demonstrate that tumors can bypass the requirement of JAM-A for myeloid cell recruitment and reprogramming.

Emerging applications of nanobodies in cancer therapy.

Cancer is a heterogeneous disease, requiring treatment tailored to the unique phenotype of the patient's tumor. Monoclonal antibodies (mAbs) and variants thereof have enabled targeted therapies to selectively target cancer cells. Cancer cell-specific mAbs have been used for image-guided surgery and targeted delivery of radionuclides or toxic agents, improving classical treatment strategies. Cancer cell-specific mAbs can further inhibit tumor cell growth or can stimulate immune-mediated destruction of cancer cells, a feature that has also been achieved through mAb-mediated manipulation of immune cells and pathways. Drawbacks of mAbs and their variants, together with the discovery of camelid heavy chain-only antibodies and the many advantageous features of their variable domains, referred to as VHHs, single domain antibodies or nanobodies (Nbs), resulted in the exploration of Nbs as an alternative targeting moiety. We therefore review the state-of-the-art as well as novel exploitation strategies of Nbs for targeted cancer therapy.

TNF-α-Secreting Lung Tumor-Infiltrated Monocytes Play a Pivotal Role During Anti-PD-L1 Immunotherapy.

Immune checkpoint blockade (ICB) of the PD-1 pathway revolutionized the survival forecast for advanced non-small cell lung cancer (NSCLC). Yet, the majority of PD-L1+ NSCLC patients are refractory to anti-PD-L1 therapy. Recent observations indicate a pivotal role for the PD-L1+ tumor-infiltrating myeloid cells in therapy failure. As the latter comprise a heterogenous population in the lung tumor microenvironment, we applied an orthotopic Lewis Lung Carcinoma (LLC) model to evaluate 11 different tumor-residing myeloid subsets in response to anti-PD-L1 therapy. While we observed significantly reduced fractions of tumor-infiltrating MHC-IIlow macrophages and monocytes, serological levels of TNF-α restored in lung tumor-bearing mice. Notably, we demonstrated in vivo and in vitro that anti-PD-L1 therapy mediated a monocyte-specific production of, and response to TNF-α, further accompanied by their significant upregulation of CD80, VISTA, LAG-3, SIRP-α and TIM-3. Nevertheless, co-blockade of PD-L1 and TNF-α did not reduce LLC tumor growth. A phenomenon that was partly explained by the observation that monocytes and TNF-α play a Janus-faced role in anti-PD-L1 therapy-mediated CTL stimulation. This was endorsed by the observation that monocytes appeared crucial to effectively boost T cell-mediated LLC killing in vitro upon combined PD-L1 with LAG-3 or SIRP-α blockade. Hence, this study enlightens the biomarker potential of lung tumor-infiltrated monocytes to define more effective ICB combination strategies.

Targeted Radionuclide Therapy with Low and High-Dose Lutetium-177-Labeled Single Domain Antibodies Induces Distinct Immune Signatures in a Mouse Melanoma Model.

Targeted radionuclide therapy (TRT) using probes labeled with Lutetium-177 (177Lu) represents a new and growing type of cancer therapy. We studied immunologic changes in response to TRT with 177Lu labeled anti-human CD20 camelid single domain antibodies (sdAb) 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 IL10 serum levels. Low-dose TRT also decreased CCL5 serum levels. At the tumor, high-dose TRT induced a type I IFN gene signature, while low-dose TRT induced a proinflammatory 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 Th1 cells in addition to double negative T cells. In conclusion, these data suggest that low and high-dose TRT induce distinct immunologic changes, which might serve as an anchoring point for combination therapy.

Inhibiting Histone and DNA Methylation Improves Cancer Vaccination in an Experimental Model of Melanoma.

Immunotherapy has improved the treatment of malignant skin cancer of the melanoma type, yet overall clinical response rates remain low. Combination therapies could be key to meet this cogent medical need. Because epigenetic hallmarks represent promising combination therapy targets, we studied the immunogenic potential of a dual inhibitor of histone methyltransferase G9a and DNA methyltransferases (DNMTs) in the preclinical B16-OVA melanoma model. Making use of tumor transcriptomic and functional analyses, methylation-targeted epigenetic reprogramming was shown to induce tumor cell cycle arrest and apoptosis in vitro coinciding with transient tumor growth delay and an IFN-I response in immune-competent mice. In consideration of a potential impact on immune cells, the drug was shown not to interfere with dendritic cell maturation or T-cell activation in vitro. Notably, the drug promoted dendritic cell and, to a lesser extent, T-cell infiltration in vivo, yet failed to sensitize tumor cells to programmed cell death-1 inhibition. Instead, it increased therapeutic efficacy of TCR-redirected T cell and dendritic cell vaccination, jointly increasing overall survival of B16-OVA tumor-bearing mice. The reported data confirm the prospect of methylation-targeted epigenetic reprogramming in melanoma and sustain dual G9a and DNMT inhibition as a strategy to tip the cancer-immune set-point towards responsiveness to active and adoptive vaccination against melanoma.

T-cell subsets in the skin and their role in inflammatory skin disorders.

T lymphocytes (T cells) are major players of the adaptive immune response. Naive T cells are primed in the presence of cytokines, leading to polarization into distinct T-cell subsets with specific functions. These subsets are classified based on their T-cell receptor profile, expression of transcription factors, surface cytokine and chemokine receptors, and their cytokine production, which together determine their specific function. This review provides an overview of the various T-cell subsets and their function in several inflammatory skin disorders ranging from allergic inflammation to skin tumors. Moreover, we highlight similarities of T-cell responses across different skin disorders, demonstrating the presence of similar and opposing functions for the different T-cell subsets. Finally, we discuss the effects of currently available and promising therapeutic approaches to harness T cells in inflammatory skin diseases for which efficacy next to unwanted side effects provide new insights into the pathophysiology of skin disorders.

Transcutaneous Vagal Nerve Stimulation Alone or in Combination With Radiotherapy Stimulates Lung Tumor Infiltrating Lymphocytes But Fails to Suppress Tumor Growth.

The combination of radiotherapy (RT) with immunotherapy represents a promising treatment modality for non-small cell lung cancer (NSCLC) patients. As only a minority of patients shows a persistent response today, a spacious optimization window remains to be explored. Previously we showed that fractionated RT can induce a local immunosuppressive profile. Based on the evolving concept of an immunomodulatory role for vagal nerve stimulation (VNS), we tested its therapeutic and immunological effects alone and in combination with fractionated RT in a preclinical-translational study. Lewis lung carcinoma-bearing C57Bl/6 mice were treated with VNS, fractionated RT or the combination while a patient cohort with locally advanced NSCLC receiving concurrent radiochemotherapy (ccRTCT) was enrolled in a clinical trial to receive either sham or effective VNS daily during their 6 weeks of ccRTCT treatment. Preclinically, VNS alone or with RT showed no therapeutic effect yet VNS alone significantly enhanced the activation profile of intratumoral CD8+ T cells by upregulating their IFN-γ and CD137 expression. In the periphery, VNS reduced the RT-mediated rise of splenic, but not blood-derived, regulatory T cells (Treg) and monocytes. In accordance, the serological levels of protumoral CXCL5 next to two Treg-attracting chemokines CCL1 and CCL22 were reduced upon VNS monotherapy. In line with our preclinical findings on the lack of immunological changes in blood circulating immune cells upon VNS, immune monitoring of the peripheral blood of VNS treated NSCLC patients (n=7) did not show any significant changes compared to ccRTCT alone. As our preclinical data do suggest that VNS intensifies the stimulatory profile of the tumor infiltrated CD8+ T cells, this favors further research into non-invasive VNS to optimize current response rates to RT-immunotherapy in lung cancer patients.

Formatting and gene-based delivery of a human PD-L1 single domain antibody for immune checkpoint blockade.

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-1POS cells than was monovalent K2. We further showed that bivalent constructs generated using a 30 GS linker or disulfide bond were 169- and 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.

Plasma zinc status and hyperinflammatory syndrome in hospitalized COVID-19 patients: An observational study.

Zinc deficiency is associated with impaired antiviral response, cytokine releasing syndrome (CRS), and acute respiratory distress syndrome. Notably, similar complications are being observed during severe SARS-CoV-2 infection. We conducted a prospective, single-center, observational study in a tertiary university hospital (CUB-Hôpital Erasme, Brussels) to address the zinc status, the association between the plasma zinc concentration, development of CRS, and the clinical outcomes in PCR-confirmed and hospitalized COVID-19 patients. One hundred and thirty-nine eligible patients were included between May 2020 and November 2020 (median age of 65 years [IQR = 54, 77]). Our cohort's median plasma zinc concentration was 57 µg/dL (interquartile range [IQR] = 45, 67) compared to 74 µg/dL (IQR = 64, 84) in the retrospective non-COVID-19 control group (N = 1513; p < 0.001). Markedly, the absolute majority of COVID-19 patients (96%) were zinc deficient (<80 µg/dL). The median zinc concentration was lower in patients with CRS compared to those without CRS (-5 µg/dL; 95% CI = -10.5, 0.051; p = 0.048). Among the tested outcomes, zinc concentration is significantly correlated with only the length of hospital stay (rho = -0.19; p = 0.022), but not with mortality or morbidity. As such, our findings do not support the role of zinc as a robust prognostic marker among hospitalized COVID-19 patients who in our cohort presented a high prevalence of zinc deficiency. It might be more beneficial to explore the role of zinc as a biomarker for assessing the risk of developing a tissue-damaging CRS and predicting outcomes in patients diagnosed with COVID-19 at the early stage of the disease.

TNFα and Immune Checkpoint Inhibition: Friend or Foe for Lung Cancer?

Tumor necrosis factor-alpha (TNFα) can bind two distinct receptors (TNFR1/2). The transmembrane form (tmTNFα) preferentially binds to TNFR2. Upon tmTNFα cleavage by the TNF-alpha-converting enzyme (TACE), its soluble (sTNFα) form is released with higher affinity for TNFR1. This assortment empowers TNFα with a plethora of opposing roles in the processes of tumor cell survival (and apoptosis) and anti-tumor immune stimulation (and suppression), in addition to angiogenesis and metastases. Its functions and biomarker potential to predict cancer progression and response to immunotherapy are reviewed here, with a focus on lung cancer. By mining existing sequencing data, we further demonstrate that the expression levels of TNF and TACE are significantly decreased in lung adenocarcinoma patients, while the TNFR1/TNFR2 balance are increased. We conclude that the biomarker potential of TNFα alone will most likely not provide conclusive findings, but that TACE could have a key role along with the delicate balance of sTNFα/tmTNFα as well as TNFR1/TNFR2, hence stressing the importance of more research into the potential of rationalized treatments that combine TNFα pathway modulators with immunotherapy for lung cancer patients.

Fractionated Radiation Severely Reduces the Number of CD8+ T Cells and Mature Antigen Presenting Cells Within Lung Tumors.

PURPOSE: The combination of standard-of-care radiation therapy (RT) with immunotherapy is moving to the mainstream of non-small cell lung cancer treatment. Multiple preclinical studies reported on the CD8+ T cell stimulating properties of RT, resulting in abscopal therapeutic effects. A literature search demonstrates that most preclinical lung cancer studies applied subcutaneous lung tumor models. Hence, in-depth immunologic evaluation of clinically relevant RT in orthotopic lung cancer models is lacking. METHODS AND MATERIALS: We studied the therapeutic and immunologic effects of low-dose fractionated RT on lungs from C57BL/6 mice, challenged 2 weeks before with firefly luciferase expressing Lewis lung carcinoma cells via the tail vein. Low-dose fractionation was represented by 4 consecutive daily fractions of image guided RT at 3.2 Gy. RESULTS: We showed reduced lung tumor growth upon irradiation using in vivo bioluminescence imaging and immunohistochemistry. Moreover, significant immunologic RT-induced changes were observed in irradiated lungs and in the periphery (spleen and blood). First, a significant decrease in the number of CD8+ T cells and trends toward more CD4+ and regulatory T cells were seen after RT in all evaluated tissues. Notably, only in the periphery did the remaining CD8+ T cells show a more activated phenotype. In addition, a significant expansion of neutrophils and monocytes was observed upon RT locally and systemically. Locally, RT increased the influx of tumor-associated macrophages and conventional type 2 dendritic cells, whereas the alveolar macrophages and conventional type 1 DCs dramatically decreased. Functionally, these antigen-presenting cells severely reduced their CD86 expression, suggesting a reduced capacity to induce potent immunity. CONCLUSIONS: Our results imply that low-dose fractionated RT of tumor-bearing lung tissue shifts the immune cell balance toward an immature myeloid cell dominating profile. These data argue for myeloid cell repolarizing strategies to enhance the abscopal effects in patients with non-small cell lung cancer treated with fractionated RT.

Single-Domain Antibody Nuclear Imaging Allows Noninvasive Quantification of LAG-3 Expression by Tumor-Infiltrating Leukocytes and Predicts Response of Immune Checkpoint Blockade.

Recent advances in the field of immune-oncology led to the discovery of next-generation immune checkpoints (ICPs). Lymphocyte activation gene-3 (LAG-3), being the most widely studied among them, is being explored as a target for the treatment of cancer patients. Several antagonistic anti-LAG-3 antibodies are being developed and are prime candidates for clinical application. Furthermore, validated therapies targeting cytotoxic T-lymphocyte-associated protein-4, programmed cell-death protein-1, or programmed cell-death ligand-1 showed that only subsets of patients respond. This finding highlights the need for better tools for patient selection and monitoring. The potential of molecular imaging to detect ICPs noninvasively in cancer is supported by several preclinical and clinical studies. Here, we report on a single-domain antibody to evaluate whole-body LAG-3 expression in various syngeneic mouse cancer models using nuclear imaging. Methods: SPECT/CT scans of tumor-bearing mice were performed 1 h after injection with radiolabeled single-domain antibody. Organs and tumors of mice were isolated and evaluated for the presence of the radiolabeled tracer and LAG-3-expressing immune cells using a γ-counter and flow cytometry respectively. PD-1/LAG-3-blocking antibodies were injected in MC38-bearing mice. Results: The radiolabeled single-domain antibody detected LAG-3 expression on tumor-infiltrating lymphocytes (TILs) as soon as 1 h after injection in MC38, MO4, and TC-1 cancer models. The single-domain antibody tracer visualized a compensatory upregulation of LAG-3 on TILs in MC38 tumors of mice treated with PD-1-blocking antibodies. When PD-1 blockade was combined with LAG-3 blockade, a synergistic effect on tumor growth delay was observed. Conclusion: These findings consolidate LAG-3 as a next-generation ICP and support the use of single-domain antibodies as tools to noninvasively monitor the dynamic evolution of LAG-3 expression by TILs, which could be exploited to predict therapy outcome.

Targeting Neuropilin-1 with Nanobodies Reduces Colorectal Carcinoma Development.

Neuropilin-1 (NRP-1) is a co-receptor for semaphorins and vascular endothelial growth factor (VEGF) family members that can be expressed on cancer cells and tumor-infiltrating myeloid, endothelial and lymphoid cells. It has been linked to a tumor-promoting environment upon interaction with semaphorin 3A (Sema3A). Nanobodies (Nbs) targeting NRP-1 were generated for their potential to hamper the NRP-1/Sema3A interaction and their impact on colorectal carcinoma (CRC) development was evaluated in vivo through the generation of anti-NRP-1-producing CRC cells. We observed that tumor growth was significantly delayed and survival prolonged when the anti-NRP-1 Nbs were produced in vivo. We further analyzed the tumor microenvironment and observed that the pro-inflammatory MHC-IIhigh/trophic MHC-IIlow macrophage ratio was increased in tumors that produce anti-NRP-1 Nbs. This finding was corroborated by an increase in the expression of genes associated with MHC-IIhigh macrophages and a decrease in the expression of MHC-IIlow macrophage-associated genes in the macrophage pool sorted from anti-NRP-1 Nb-producing tumors. Moreover, we observed a significantly higher percentage of tumor-associated antigen-specific CD8+ T cells in tumors producing anti-NRP-1 Nbs. These data demonstrate that an intratumoral expression of NRP-1/Sema3A blocking biologicals increases anti-tumor immunity.

Hepatocarcinoma Induces a Tumor Necrosis Factor-Dependent Kupffer Cell Death Pathway That Favors Its Proliferation Upon Partial Hepatectomy.

Partial hepatectomy (PH) is the main treatment for early-stage hepatocellular carcinoma (HCC). Yet, a significant number of patients undergo recursion of the disease that could be linked to the fate of innate immune cells during the liver regeneration process. In this study, using a murine model, we investigated the impact of PH on HCC development by bioluminescence imaging and flow cytometry. While non-resected mice were able to control and reject orthotopic implanted Hepa1-6 hepatocarcinoma cells, resected liver underwent an increased tumoral proliferation. This phenomenon was associated with a PH-induced reduction in the number of liver-resident macrophages, i.e., Kupffer cells (KC). Using a conditional ablation model, KC were proved to participate in Hepa1-6 rejection. We demonstrated that in the absence of Hepa1-6, PH-induced KC number reduction was dependent on tumor necrosis factor-alpha (TNF-α), receptor-interacting protein kinase (RIPK) 3, and caspase-8 activation, whereas interleukin (IL)-6 acted as a KC pro-survival signal. In mice with previous Hepa1-6 encounter, the KC reduction switched toward a TNF-α-RIPK3-caspase-1 activation. Moreover, KC disappearance associated with caspase-1 activity induced the recruitment of monocyte-derived cells that are beneficial for tumor growth, while caspase-8-dependent reduction did not. In conclusion, our study highlights the importance of the TNF-α-dependent death pathway induced in liver macrophages following partial hepatectomy in regulating the antitumoral immune responses.

Theranostics in immuno-oncology using nanobody derivatives.

Targeted therapy and immunotherapy have become mainstream in cancer treatment. However, only patient subsets benefit from these expensive therapies, and often responses are short-lived or coincide with side effects. A growing modality in precision oncology is the development of theranostics, as this enables patient selection, treatment and monitoring. In this approach, labeled compounds and an imaging technology are used to diagnose patients and select the best treatment option, whereas for therapy, related compounds are used to target cancer cells or the tumor stroma. In this context, nanobodies and nanobody-directed therapeutics have gained interest. This interest stems from their high antigen specificity, small size, ease of labeling and engineering, allowing specific imaging and design of therapies targeting antigens on tumor cells, immune cells as well as proteins in the tumor environment. This review provides a comprehensive overview on the state-of-the-art regarding the use of nanobodies as theranostics, and their importance in the emerging field of personalized medicine.