Generation and Characterization of Novel Pan-Cancer Anti-uPAR Fluorescent Nanobodies as Tools for Image-Guided Surgery.

Fluorescence molecular imaging plays a vital role in image-guided surgery. In this context, the urokinase plasminogen activator receptor (uPAR) is an interesting biomarker enabling the detection and delineation of various tumor types due to its elevated expression on both tumor cells and the tumor microenvironment. In this study, anti-uPAR Nanobodies (Nbs) are generated through llama immunization with human and murine uPAR protein. Extensive in vitro characterization and in vivo testing with radiolabeled variants are conducted to assess their pharmacokinetics and select lead compounds. Subsequently, the selected Nbs are converted into fluorescent agents, and their application for fluorescence-guided surgery is evaluated in various subcutaneous and orthotopic tumor models. The study yields a panel of high-affinity anti-uPAR Nbs, showing specific binding across multiple types of cancer cells in vitro and in vivo. Lead fluorescently-labeled compounds exhibit high tumor uptake with high contrast at 1 h after intravenous injection across all assessed uPAR-expressing tumor models, outperforming a non-targeting control Nb. Additionally, rapid and accurate tumor localization and demarcation are demonstrated in an orthotopic human glioma model. Utilizing these Nbs can potentially enhance the precision of surgical tumor resection and, consequently, improve survival rates in the clinic.

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.

Alpha-galactosylceramide improves the potency of mRNA LNP vaccines against cancer and intracellular bacteria.

Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8+ T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8+ T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12-200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections.

AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in Acute Myeloid Leukemia.

Rationale: AXL expression has been identified as a prognostic factor in acute myeloid leukemia (AML) and is detectable in approximately 50% of AML patients. In this study, we developed AXL-specific single domain antibodies (sdAbs), cross-reactive for both mouse and human AXL protein, to non-invasively image and treat AXL-expressing cancer cells. Methods: AXL-specific sdAbs were induced by immunizing an alpaca with mouse and human AXL proteins. SdAbs were characterized using ELISA, flow cytometry, surface plasmon resonance and the AlphaFold2 software. A lead compound was selected and labeled with 99mTc for evaluation as a diagnostic tool in mouse models of human (THP-1 cells) or mouse (C1498 cells) AML using SPECT/CT imaging. For therapeutic purposes, the lead compound was fused to a mouse IgG2a-Fc tail and in vitro functionality tests were performed including viability, apoptosis and proliferation assays in human AML cell lines and primary patient samples. Using these in vitro models, its anti-tumor effect was evaluated as a single agent, and in combination with standard of care agents venetoclax or cytarabine. Results: Based on its cell binding potential, cross-reactivity, nanomolar affinity and GAS6/AXL blocking capacity, we selected sdAb20 for further evaluation. Using SPECT/CT imaging, we observed tumor uptake of 99mTc-sdAb20 in mice with AXL-positive THP-1 or C1498 tumors. In THP-1 xenografts, an optimized protocol using pre-injection of cold sdAb20-Fc was required to maximize the tumor-to-background signal. Besides its diagnostic value, we observed a significant reduction in tumor cell proliferation and viability using sdAb20-Fc in vitro. Moreover, combining sdAb20-Fc and cytarabine synergistically induced apoptosis in human AML cell lines, while these effects were less clear when combined with venetoclax. Conclusions: Because of their diagnostic potential, sdAbs could be used to screen patients eligible for AXL-targeted therapy and to follow-up AXL expression during treatment and disease progression. When fused to an Fc-domain, sdAbs acquire additional therapeutic properties that can lead to a multidrug approach for the treatment of AXL-positive cancer patients.

Assessment of Connexin43 Hemichannel Functionality Based on Cytosolic Uptake of Yo-Pro1.

Connexin proteins are the building blocks of gap junctions and connexin hemichannels. Both provide a pathway for cellular communication. Gap junctions support intercellular communication mechanisms and regulate homeostasis. In contrast, open connexin hemichannels connect the intracellular compartment and the extracellular environment, and their activation fuels inflammation and cell death. The development of clinically applicable connexin hemichannel blockers for therapeutic purposes is therefore gaining momentum. This chapter describes a well-established protocol optimized for assessing connexin hemichannel activity by using the reporter dye Yo-Pro1.

Novel technologies for applying immune checkpoint blockers.

Cancer cells develop several ways to subdue the immune system among others via upregulation of inhibitory immune checkpoint (ICP) proteins. These ICPs paralyze immune effector cells and thereby enable unfettered tumor growth. Monoclonal antibodies (mAbs) that block ICPs can prevent immune exhaustion. Due to their outstanding effects, mAbs revolutionized the field of cancer immunotherapy. However, current ICP therapy regimens suffer from issues related to systemic administration of mAbs, including the onset of immune related adverse events, poor pharmacokinetics, limited tumor accessibility and immunogenicity. These drawbacks and new insights on spatiality prompted the exploration of novel administration routes for mAbs for instance peritumoral delivery. Moreover, novel ICP drug classes that are adept to novel delivery technologies were developed to circumvent the drawbacks of mAbs. We therefore review the state-of-the-art and novel delivery strategies of ICP drugs.

Sustained release of a human PD-L1 single-domain antibody using peptide-based hydrogels.

Monoclonal antibodies (mAbs) targeting the immune checkpoint axis, which contains the programmed cell death protein-1 (PD-1) and its ligand PD-L1, revolutionized the field of oncology. Unfortunately, the large size of mAbs and the presence of an Fc fraction limit their tumor penetrative capacities and support off-target effects, potentially resulting in unresponsive patients and immune-related adverse events (irAEs) respectively. Single-domain antibodies (sdAbs) are ten times smaller than conventional mAbs and represent an emerging antibody subclass that has been proposed as next generation immune checkpoint inhibitor (ICI) therapeutics. They demonstrate favorable characteristics, such as an excellent stability, high antigen-binding affinity and an enhanced tumor penetration. Because sdAbs have a short half-life, methods to prolong their presence in the circulation and at the target site might be necessary in some cases to unfold their full therapeutic potential. In this study, we investigated a peptide-based hydrogel as an injectable biomaterial depot formulation for the sustained release of the human PD-L1 sdAb K2. We showed that a hydrogel composed of the amphipathic hexapeptide hydrogelator H-FQFQFK-NH2 prolonged the in vivo release of K2 after subcutaneous (s.c.) injection, up to at least 72 h, as monitored by SPECT/CT and fluorescence imaging. Additionally, after encapsulation in the hydrogel and s.c. administration, a significantly extended systemic presence and tumor uptake of K2 was observed in mice bearing a melanoma tumor expressing human PD-L1. Altogether, this study describes how peptide hydrogels can be exploited to provide the sustained release of sdAbs, thereby potentially enhancing its clinical and therapeutic effects.

Colorectal Cancer Immunotherapy: State of the Art and Future Directions.

Cancer immunotherapy has become an indispensable mode of treatment for a multitude of solid tumor cancers. Colorectal cancer (CRC) has been one of the many cancer types to benefit from immunotherapy, especially in advanced disease where standard treatment fails to prevent recurrence or results in poor survival. The efficacy of immunotherapy in CRC has not been without challenge, as early clinical trials observed dismal responses in unselected CRC patients treated with checkpoint inhibitors. Many studies and clinical trials have since refined immunotherapies available for CRC, solidifying immunotherapy as a powerful asset for CRC treatment. This review article examines CRC immunotherapies, from their foundation, through emerging avenues for improvement, to future directions.

Targeting S100A9 protein affects mTOR-ER stress signaling and increases venetoclax sensitivity in Acute Myeloid Leukemia.

Acute Myeloid Leukemia (AML) is a heterogeneous disease with limited treatment options and a high demand for novel targeted therapies. Since myeloid-related protein S100A9 is abundantly expressed in AML, we aimed to unravel the therapeutic impact and underlying mechanisms of targeting both intracellular and extracellular S100A9 protein in AML cell lines and primary patient samples. S100A9 silencing in AML cell lines resulted in increased apoptosis and reduced AML cell viability and proliferation. These therapeutic effects were associated with a decrease in mTOR and endoplasmic reticulum stress signaling. Comparable results on AML cell proliferation and mTOR signaling could be observed using the clinically available S100A9 inhibitor tasquinimod. Interestingly, while siRNA-mediated targeting of S100A9 affected both extracellular acidification and mitochondrial metabolism, tasquinimod only affected the mitochondrial function of AML cells. Finally, we found that S100A9-targeting approaches could significantly increase venetoclax sensitivity in AML cells, which was associated with a downregulation of BCL-2 and c-MYC in the combination group compared to single agent therapy. This study identifies S100A9 as a novel molecular target to treat AML and supports the therapeutic evaluation of tasquinimod in venetoclax-based regimens for AML patients.

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.

Development and evaluation of nanobody tracers for noninvasive nuclear imaging of the immune-checkpoint TIGIT.

Introduction: T cell Ig and ITIM domain receptor (TIGIT) is a next-generation immune checkpoint predominantly expressed on activated T cells and NK cells, exhibiting an unfavorable prognostic association with various malignancies. Despite the emergence of multiple TIGIT-blocking agents entering clinical trials, only a fraction of patients responded positively to anti-TIGIT therapy. Consequently, an urgent demand arises for noninvasive techniques to quantify and monitor TIGIT expression, facilitating patient stratification and enhancing therapeutic outcomes. Small antigen binding moieties such as nanobodies, are promising candidates for such tracer development. Methods: We generated a panel of anti-human or anti-mouse TIGIT nanobodies from immunized llamas. In addition, we designed a single-chain variable fragment derived from the clinically tested monoclonal antibody Vibostolimab targeting TIGIT, and assessed its performance alongside the nanobodies. In vitro characterization studies were performed, including binding ability and affinity to cell expressed or recombinant TIGIT. After Technetium-99m labeling, the nanobodies and the single-chain variable fragment were evaluated in vivo for their ability to detect TIGIT expression using SPECT/CT imaging, followed by ex vivo biodistribution analysis. Results: Nine nanobodies were selected for binding to recombinant and cell expressed TIGIT with low sub-nanomolar affinities and are thermostable. A six-fold higher uptake in TIGIT-overexpressing tumor was demonstrated one hour post- injection with Technetium-99m labeled nanobodies compared to an irrelevant control nanobody. Though the single-chain variable fragment exhibited superior binding to TIGIT-expressing peripheral blood mononuclear cells in vitro, its in vivo behavior yielded lower tumor-to-background ratios at one hour post- injection, indicating that nanobodies are better suited for in vivo imaging than the single-chain variable fragment. Despite the good affinity, high specificity and on-target uptake in mice in this setting, imaging of TIGIT expression on tumor- infiltrating lymphocytes within MC38 tumors remained elusive. This is likely due to the low expression levels of TIGIT in this model. Discussion: The excellent affinity, high specificity and rapid on-target uptake in mice bearing TIGIT- overexpressing tumors showed the promising diagnostic potential of nanobodies to noninvasively image high TIGIT expression within the tumor. These findings hold promise for clinical translation to aid patient selection and improve therapy response.

Recent Advances Using Genetic Therapies Against Infectious Diseases and for Vaccination.

The development of prophylatic or therapeutic medicines for infectious diseases is one of the priorities for health organizations worldwide. Innovative solutions are required to achieve effective, safe, and accessible treatments for most if not all infectious diseases, particularly those that are chronic in nature or that emerge unexpectedly over time. Genetic technologies offer versatile possibilities to design therapies against pathogens. Recent developments such as mRNA vaccines, CRISPR gene editing, and immunotherapies provide unprecedented hope to achieve significant results in the field of infectious diseases. This review will focus on advances in this domain, showcasing the cross-fertilization with other fields (e.g., oncology), and addressing some of the logistical and economic concerns important to consider when making these advances accessible to diverse populations around the world.

Phase I Study of [68Ga]Ga-Anti-CD206-sdAb for PET/CT Assessment of Protumorigenic Macrophage Presence in Solid Tumors (MMR Phase I).

Macrophages play an important role throughout the body. Antiinflammatory macrophages expressing the macrophage mannose receptor (MMR, CD206) are involved in disease development, ranging from oncology to atherosclerosis and rheumatoid arthritis. [68Ga]Ga-NOTA-anti-CD206 single-domain antibody (sdAb) is a PET tracer targeting CD206. This first-in-human study, as its primary objective, evaluated the safety, biodistribution, and dosimetry of this tracer. The secondary objective was to assess its tumor uptake. Methods: Seven patients with a solid tumor of at least 10 mm, an Eastern Cooperative Oncology Group score of 0 or 1, and good renal and hepatic function were included. Safety was evaluated using clinical examination and blood sampling before and after injection. For biodistribution and dosimetry, PET/CT was performed at 11, 90, and 150 min after injection; organs showing tracer uptake were delineated, and dosimetry was evaluated. Blood samples were obtained at selected time points for blood clearance. Metabolites in blood and urine were assessed. Results: Seven patients were injected with, on average, 191 MBq of [68Ga]Ga-NOTA-anti-CD206-sdAb. Only 1 transient adverse event of mild severity was considered to be possibly, although unlikely, related to the study drug (headache, Common Terminology Criteria for Adverse Events grade 1). The blood clearance was fast, with less than 20% of the injected activity remaining after 80 min. There was uptake in the liver, kidneys, spleen, adrenals, and red bone marrow. The average effective dose from the radiopharmaceutical was 4.2 mSv for males and 5.2 mSv for females. No metabolites were detected. Preliminary data of tumor uptake in cancer lesions showed higher uptake in the 3 patients who subsequently progressed than in the 3 patients without progression. One patient could not be evaluated because of technical failure. Conclusion: [68Ga]Ga-NOTA-anti-CD206-sdAb is safe and well tolerated. It shows rapid blood clearance and renal excretion, enabling high contrast-to-noise imaging at 90 min after injection. The radiation dose is comparable to that of routinely used PET tracers. These findings and the preliminary results in cancer patients warrant further investigation of this tracer in phase II clinical trials.

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.

An mRNA mix redirects dendritic cells towards an antiviral program, inducing anticancer cytotoxic stem cell and central memory CD8+ T cells.

Dendritic cell (DC)-maturation stimuli determine the potency of these antigen-presenting cells and, therefore, the quality of the T-cell response. Here we describe that the maturation of DCs via TriMix mRNA, encoding CD40 ligand, a constitutively active variant of toll-like receptor 4 and the co-stimulatory molecule CD70, enables an antibacterial transcriptional program. Besides, we further show that the DCs are redirected into an antiviral transcriptional program when CD70 mRNA in TriMix is replaced with mRNA encoding interferon-gamma and a decoy interleukin-10 receptor alpha, forming a four-component mixture referred to as TetraMix mRNA. The resulting TetraMixDCs show a high potential to induce tumor antigen-specific T cells within bulk CD8+ T cells. Tumor-specific antigens (TSAs) are emerging and attractive targets for cancer immunotherapy. As T-cell receptors recognizing TSAs are predominantly present on naive CD8+ T cells (TN), we further addressed the activation of tumor antigen-specific T cells when CD8+ TN cells are stimulated by TriMixDCs or TetraMixDCs. In both conditions, the stimulation resulted in a shift from CD8+ TN cells into tumor antigen-specific stem cell-like memory, effector memory and central memory T cells with cytotoxic capacity. These findings suggest that TetraMix mRNA, and the antiviral maturation program it induces in DCs, triggers an antitumor immune reaction in cancer patients.

Tasquinimod suppresses tumor cell growth and bone resorption by targeting immunosuppressive myeloid cells and inhibiting c-MYC expression in multiple myeloma.

BACKGROUND: Immunotherapy emerged as a promising treatment option for multiple myeloma (MM) patients. However, therapeutic efficacy can be hampered by the presence of an immunosuppressive bone marrow microenvironment including myeloid cells. S100A9 was previously identified as a key regulator of myeloid cell accumulation and suppressive activity. Tasquinimod, a small molecule inhibitor of S100A9, is currently in a phase Ib/IIa clinical trial in MM patients (NCT04405167). We aimed to gain more insights into its mechanisms of action both on the myeloma cells and the immune microenvironment. METHODS: We analyzed the effects of tasquinimod on MM cell viability, cell proliferation and downstream signaling pathways in vitro using RNA sequencing, real-time PCR, western blot analysis and multiparameter flow cytometry. Myeloid cells and T cells were cocultured at different ratios to assess tasquinimod-mediated immunomodulatory effects. The in vivo impact on immune cells (myeloid cell subsets, macrophages, dendritic cells), tumor load, survival and bone disease were elucidated using immunocompetent 5TMM models. RESULTS: Tasquinimod treatment significantly decreased myeloma cell proliferation and colony formation in vitro, associated with an inhibition of c-MYC and increased p27 expression. Tasquinimod-mediated targeting of the myeloid cell population resulted in increased T cell proliferation and functionality in vitro. Notably, short-term tasquinimod therapy of 5TMM mice significantly increased the total CD11b+ cells and shifted this population toward a more immunostimulatory state, which resulted in less myeloid-mediated immunosuppression and increased T cell activation ex vivo. Tasquinimod significantly reduced the tumor load and increased the trabecular bone volume, which resulted in prolonged overall survival of MM-bearing mice in vivo. CONCLUSION: Our study provides novel insights in the dual therapeutic effects of the immunomodulator tasquinimod and fosters its evaluation in combination therapy trials for MM patients.

Challenges in neoantigen-directed therapeutics.

A fundamental prerequisite for the efficacy of cancer immunotherapy is the presence of functional, antigen-specific T cells within the tumor. Neoantigen-directed therapy is a promising strategy that aims at targeting the host's immune response against tumor-specific antigens, thereby eradicating cancer cells. Initial forays have been made in clinical environments utilizing vaccines and adoptive cell therapy; however, many challenges lie ahead. We provide an in-depth overview of the current state of the field with an emphasis on in silico neoantigen discovery and the clinical aspects that need to be addressed to unlock the full potential of this therapy.

Assessment of Human SARS CoV-2-Specific T-Cell Responses Elicited In Vitro by New Computationally Designed mRNA Immunogens (COVARNA).

The COVID-19 pandemic has brought significant changes and advances in the field of vaccination, including the implementation and widespread use of encapsidated mRNA vaccines in general healthcare practice. Here, we present two new mRNAs expressing antigenic parts of the SARS-CoV-2 spike protein and provide data supporting their functionality. The first mRNA, called RBD-mRNA, encodes a trimeric form of the virus spike protein receptor binding domain (RBD). The other mRNA, termed T-mRNA, codes for the relevant HLA I and II spike epitopes. The two mRNAs (COVARNA mRNAs) were designed to be used for delivery to cells in combination, with the RBD-mRNA being the primary source of antigen and the T-mRNA working as an enhancer of immunogenicity by supporting CD4 and CD8 T-cell activation. This innovative approach substantially differs from other available mRNA vaccines, which are largely directed to antibody production by the entire spike protein. In this study, we first show that both mRNAs are functionally transfected into human antigen-presenting cells (APCs). We obtained peripheral blood mononuclear cell (PBMC) samples from three groups of voluntary donors differing in their immunity against SARS-CoV-2: non-infected (naïve), infected-recovered (convalescent), and vaccinated. Using an established method of co-culturing autologous human dendritic cells (hDCs) with T-cells, we detected proliferation and cytokine secretion, thus demonstrating the ability of the COVARNA mRNAs to activate T-cells in an antigen-specific way. Interestingly, important differences in the intensity of the response between the infected-recovered (convalescent) and vaccinated donors were observed, with the levels of T-cell proliferation and cytokine secretion (IFNγ, IL-2R, and IL-13) being higher in the vaccinated group. In summary, our data support the further study of these mRNAs as a combined approach for future use as a vaccine.

The prognostic value and therapeutic targeting of myeloid-derived suppressor cells in hematological cancers.

The success of immunotherapeutic approaches in hematological cancers is partially hampered by the presence of an immunosuppressive microenvironment. Myeloid-derived suppressor cells (MDSC) are key components of this suppressive environment and are frequently associated with tumor cell survival and drug resistance. Based on their morphology and phenotype, MDSC are commonly subdivided into polymorphonuclear MDSC (PMN-MDSC or G-MDSC) and monocytic MDSC (M-MDSC), both characterized by their immunosuppressive function. The phenotype, function and prognostic value of MDSC in hematological cancers has been intensively studied; however, the therapeutic targeting of this cell population remains challenging and needs further investigation. In this review, we will summarize the prognostic value of MDSC and the different attempts to target MDSC (or subtypes of MDSC) in hematological cancers. We will discuss the benefits, challenges and opportunities of using MDSC-targeting approaches, aiming to enhance anti-tumor immune responses of currently used cellular and non-cellular immunotherapies.