Target Antigen-presenting Cells Research Articles

Development of a Versatile Cancer Vaccine Format Targeting Antigen-Presenting Cells Using Proximity-Based Sortase A-Mediated Ligation of T-Cell Epitopes.

Cancer vaccines are a promising strategy to increase tumor-specific immune responses in patients who do not adequately respond to checkpoint inhibitors. Cancer vaccines that contain patient-specific tumor antigens are most effective but also necessitate the production of patient-specific vaccines. This study aims to develop a versatile cancer vaccine format in which patient-specific tumor antigens can be site-specifically conjugated by a proximity-based Sortase A (SrtA)-mediated ligation (PBSL) approach to antibodies that specifically bind to antigen-presenting cells to stimulate immune responses. DEC205 and CD169 are both receptors expressed on antigen-presenting cells that can be targeted to deliver antigens and stimulate T-cell responses. We used the CRISPR/HDR platform to produce mouse heavy chain IgG2a antibodies with DEC205 or CD169 specificity containing an SrtA recognition motif followed by a SpyTag at the C-terminus. Using a recombinant protein of SrtA linked to SpyCatcher, we applied proximity-based SrtA-mediated ligation to ligate fluorescein isothiocyanate (FITC)-labeled or antigenic peptides to the antibodies. Ligated antibodies bound to DEC205-expressing dendritic cells or CD169-expressing macrophages both in vitro and in vivo. More importantly, immunization with DEC205- or CD169-specific Abs linked to T-cell epitopes efficiently stimulated T-cell responses in vivo. To conclude, we have developed a cancer vaccine format using PBSL that enables the rapid incorporation of tumor antigens and could potentially be implemented for the synthesis of personalized cancer vaccines.

Abstract LB068: Repurposing chimeric antigen receptor regulatory T cells for treating cancer

Abstract Solid tumors are the deadliest form of cancer. Over the past decade, chimeric antigen receptor (CAR) T-cell therapy has led to remarkable regression rates in leukemia and lymphoma. However, solid tumors have remained refractory to CAR T-cell therapy, with CAR T cells either failing to penetrate the solid tumor microenvironment (TME) or becoming exhausted once in it. Mechanisms behind these properties of the TME include nutrient scarcity, aberrant vascularization, stromal cell deposition, expression of inhibitory immune receptors by cancer cells, and the presence of immunosuppressive cells. Interestingly, there is a subset of T cells found in the TME - regulatory T cells (Tregs). Dedicated to suppressing immune responses, Tregs constitute a barrier to anticancer immunity by migrating to and heavily accumulating in solid tumors, inhibiting local immunity. Tregs can be redirected towards any cell surface antigen using a CAR and have been shown to be cytotoxic towards target antigen presenting cells. Can Tregs be converted from tumor protectors to anti-tumor effector cells? To test the anti-tumor potential of CAR Tregs, we modified human Tregs with an anti-CD19 CD28-CD3z CAR. Tregs activated via the CAR or endogenous TCR/CD28 were evaluated at the functional and gene expression levels. CAR and endogenous TCR/CD28 activation resulted in similar cell expansion and Treg stability (FOXP3 and HELIOS expression). However, CAR-mediated activation markedly increased Treg proinflammatory cytokine production. Moreover, compared to their TCR/CD28-activated counterparts, CAR Tregs displayed increased cytotoxic activity towards tumor cells and a diminished capacity to modulate antigen-presenting cells and suppress T cell proliferation, suggesting a global shift from suppression to inflammation. Strikingly, CAR Tregs suppressed tumor growth in immunodeficient mice inoculated with antigen-expressing B-cell leukemia, myeloid leukemia, and lung epithelial carcinoma. Single-cell RNA sequencing identified a cell cluster enriched in CAR Tregs characterized by high expression of effector T cell genes, such as IFNG, GZMB, and CD40LG, revealing that CAR activation creates a highly active population of proinflammatory Tregs. Multiplex cytokine analysis, whole-transcriptome RNA-seq, and intracellular cytokine staining confirmed that IFNG production by FOXP3+ Tregs is induced specifically by CAR activation. Of note, murine CAR Tregs are also cytotoxic towards tumor cells. Current experiments are focused on pinpointing and validating transcription factors and surface markers characteristic of proinflammatory CAR Tregs and further assessing CAR Treg antitumor efficacy in vivo. In conclusion, CAR-mediated activation induces a novel proinflammatory Treg phenotype that can be exploited in the next generation of engineered immune cell therapies against solid tumors. Citation Format: Leonardo M. Ferreira, Russell W. Cochrane, Rob A. Robino, Bryan Granger, Eva Allen, Vishnu Gudivada, Aguirre A. de Cubas, Stefano Berto. Repurposing chimeric antigen receptor regulatory T cells for treating cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB068.

A vaccine targeting antigen-presenting cells through CD40 induces protective immunity against Nipah disease

Nipah virus (NiV) has been recently ranked by the World Health Organization as being among the top eight emerging pathogens likely to cause major epidemics, whereas no therapeutics or vaccines have yet been approved. We report a method to deliver immunogenic epitopes from NiV through the targeting of the CD40 receptor of antigen-presenting cells by fusing a selected humanized anti-CD40 monoclonal antibody to the Nipah glycoprotein with conserved NiV fusion and nucleocapsid peptides. In the African green monkey model, CD40.NiV induces specific immunoglobulin A (IgA) and IgG as well as cross-neutralizing responses against circulating NiV strains and Hendra virus and Tcell responses. Challenge experiments using a NiV-B strain demonstrate the high protective efficacy of the vaccine, with all vaccinated animals surviving and showing no significant clinical signs or virus replication, suggesting that the CD40.NiV vaccine conferred sterilizing immunity. Overall, results obtained with the CD40.NiV vaccine are highly promising in terms of the breadth and efficacy against NiV.

IMPACTing autoimmunity through dual targeting of antigen presenting cells and T cells via inhibitory receptor agonism

Abstract Autoimmune diseases are triggered by a critical break in peripheral tolerance, thereby allowing the immune system to target and destroy host tissues. Tolerance mechanisms, such as inhibitory receptor (IRs) on T cells, can greatly influence autoimmune disease progression and outcomes, representing an opportunity for therapeutic intervention. Indeed, evidence in both animal models and patients suggests that IRs play a major role in limiting autoimmunity. Immunotherapies targeting IRs have revolutionized the treatment of many cancers and provided evidence that IR modulating therapeutics can significantly impact patient outcomes. However importantly, analogous IR-targeted autoimmune therapeutics have just begun to be tested. Despite interest, IR agonist antibodies have proven difficult to generate. Optimal agonist antibodies require IR superclustering, which is not efficiently induced by Fc-null antibodies. Successful agonists will likely rely on simultaneous Fc (constant region) tethering on antigen presenting cells (APC), thereby allowing efficient IR superclustering and downstream signaling. Current agonistic antibodies targeting IRs contain an IgG1 wild-type Fc, which by binding both activating and inhibitory Fc receptors can trigger unwanted production of inflammatory cytokines by APCs. Here we show that antibodies binding selectively FcγRIIb, support superior agonism compared to IgG1 wild-type antibodies and mitigate potential liability of current IR agonists under clinical investigation. Indeed, dual targeting of the APC and T cells shows promising pre-clinical results and will be used to create novel IR agonists.

Abstract CT274: Individualized APC targeting VB10.NEO cancer vaccines induce broad neoepitope-specific CD8 T cell responses in patients with advanced or metastatic solid tumors: interim results from a phase 1/2a trial

Abstract Background: VB N-01 is an open label phase 1/2a basket trial to evaluate safety, feasibility, and immunogenicity of a therapeutic DNA cancer vaccine VB10.NEO in patients with locally advanced or metastatic solid cancers. Each VB10.NEO vaccine contains up to 20 neoepitopes selected by the proprietary AI platform NeoSELECT and is designed to target antigen presenting cells using Nykode’ s modular vaccine platform known as Vaccibody™. Patients and Methods: The trial enrolled patients with locally advanced or metastatic solid cancers (renal cell carcinoma, urothelial cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, and melanoma), who did not obtain complete responses on immune checkpoint inhibitor therapy (CPI). Up to 14 VB10.NEO doses (3 mg per dose) were administered i.m. by PharmaJet Stratis® for up to 1 year in combination with standard of care (CPI and/or other anti-cancer therapies). Blood samples and tumor biopsies were collected at baseline and during treatment for evaluation of immune responses. Results: At cut-off of May 2022, 41 patients had received at least one vaccination with VB10.NEO. The vaccine was safe and well-tolerated with no new or additional toxicity reported beyond that expected for CPIs alone. All patients displayed immune responses to a minimum of 3 neoepitopes (average 53% of vaccine neoepitopes), assessed by in vitro stimulation (IVS) interferon-gamma ELISpot. T cell responses were elicited in both high and low tumor mutational burden patients (TMB range 2-69mut/Mb). Multiple vaccinations increased the breadth and magnitude of the immune responses with vaccine-induced T cell responses (de novo and/or amplified pre-existing) measured in 95% of eligible patients. IVS intracellular cytokine staining of selected patients demonstrated that the majority of neoepitopes induced polyfunctional CD8 T cells. TCR sequencing of baseline and on-treatment samples (6 patients) showed expansion of T cell clones in both blood and tumor with selected clones being expanded in both compartments. Conclusions: VB10.NEO was generally well tolerated in patients with various pre-treated and advanced cancers. Assessment of neoepitope-specific T cell reactivity demonstrated VB10.NEO-induced broad and long-lasting T cell responses, and the majority of tested neoepitopes activated polyfunctional CD8 T cells. Pre- and post-vaccination TCRseq analysis of blood and tumor samples demonstrated the presence of blood-expanded clones in the on-treatment tumor sample potentially indicating trafficking of VB10.NEO-expanded T cells to the tumor site. Citation Format: Jürgen Krauss, Angela Krakhardt, Stephan Eisenmann, Sebastian Ochsenreither, Kaja C.G Berg, Kushi Kushekhar, Lars-Egil Fallang, Mikkel W. Pedersen, Siri Torhaug, Karsten B. Slot, Karoline Schjetne. Individualized APC targeting VB10.NEO cancer vaccines induce broad neoepitope-specific CD8 T cell responses in patients with advanced or metastatic solid tumors: interim results from a phase 1/2a trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT274.

Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza

Conventional influenza vaccines focus on hemagglutinin (HA). However, antibody responses to neuraminidase (NA) have been established as an independent correlate of protection. Here, we introduced the ectodomain of NA into DNA vaccines that, as translated dimeric vaccine proteins, target antigen-presenting cells (APCs). The targeting was mediated by an single-chain variable fragment specific for major histocompatibility complex (MHC) class II, which is genetically linked to NA via a dimerization motif. A single immunization of BALB/c mice elicited strong and long-lasting NA-specific antibodies that inhibited NA enzymatic activity and reduced viral replication. Vaccine-induced NA immunity completely protected against a homologous influenza virus and out-competed NA immunity induced by a conventional inactivated virus vaccine. The protection was mainly mediated by antibodies, although NA-specific Tcells also contributed. APC-targeting and antigen bivalency were crucial for vaccine efficacy. The APC-targeted vaccine was potent at low doses of DNA, indicating a dose-sparing effect. Similar results were obtained with NA vaccines that targeted different surface molecules on dendritic cells. Interestingly, the protective efficacy of NA as antigen compared favorably with HA and therefore ought to receive more attention in influenza vaccine research.

The immunological effects of intradermal particle-based vaccine delivery using a novel microinjection needle studied in a human skin explant model.

For intradermal (ID) immunisation, novel needle-based delivery systems have been proposed as a better alternative to the Mantoux method. However, the penetration depth of needles in the human skin and its effect on immune cells residing in the different layers of the skin has not been analyzed. A novel and user-friendly silicon microinjection needle (Bella-muTM) has been developed, which allows for a perpendicular injection due to its short needle length (1.4-1.8mm) and ultrashort bevel. We aimed to characterize the performance of this microinjection needle in the context of the delivery of a particle-based outer membrane vesicle (OMV) vaccine using an ex vivo human skin explant model. We compared the needles of 1.4 and 1.8mm with the conventional Mantoux method to investigate the depth of vaccine injection and the capacity of the skin antigen-presenting cell (APC) to phagocytose the OMVs. The 1.4mm needle deposited the antigen closer to the epidermis than the 1.8mm needle or the Mantoux method. Consequently, activation of epidermal Langerhans cells was significantly higher as determined by dendrite shortening. We found that five different subsets of dermal APCs are able to phagocytose the OMV vaccine, irrespective of the device or injection method. ID delivery using the 1.4mm needle of a OMV-based vaccine allowed epidermal and dermal APC targeting, with superior activation of Langerhans cells. This study indicates that the use of a microinjection needle improves the delivery of vaccines in the human skin.

A hierarchical tumor-targeting strategy for eliciting potent antitumor immunity against triple negative breast cancer

Triple negative breast cancer (TNBC) as a highly aggressive and metastatic malignancy lacks targeting therapies nowadays. Moreover, although immune checkpoint blockade (ICB) is known to trigger anti-tumor immune response, most TNBC falls into the immunologically “cold” category unsuitable for ICB therapy due to insufficient lymphocyte infiltration. Herein, we develop a hierarchical targeting strategy for preparing a core-shell-structural nanodrug to concurrently block the programmed death ligand 1 (PD-L1) and deliver a stimulator of interferon gene (STING) agonist into tumor-infiltrating antigen-presenting cells (APCs). The nanodrug complexed the interferon stimulatory DNA (ISD) for STING activation in its core, conjugated PD-L1 antibody (aPD-L1) on its shell through a matrix metalloproteinase-2 (MMP-2) substrate peptide, and incorporated “hidden” mannose in its sublayer. Through aPD-L1-mediated active targeting of tumor cells and tumor-infiltrating APCs, the nanodrug efficiently accumulated in tumor sites. Then, the PD-L1-conjugating peptide was cleaved by tumor-enriched MMP-2, leaving aPD-L1 on target cells for ICB while exposing mannose to mediate targeted delivery of ISD into tumor-infiltrating dendritic cells (DCs) and tumor-associated macrophages (TAMs). Activating the STING signaling in DCs and TAMs not only stimulated the APCs maturation to prime anti-tumor immunity but also induced their chemokine secretion to promote tumor infiltration of anti-tumor effector T cells, thus sensitizing TNBC to the ICB therapy. Consequently, a potent antitumor immunity was evoked to effectively inhibit the tumor growth and metastasis in mice bearing orthotopic 4T1 breast cancer, showing the great potential in treating immunologically “cold” tumors.

Investigating the relationship between cytotoxic T lymphocyte mechanical output and metabolism.

In order to execute their cytolytic functions, cytotoxic T lymphocytes (CTLs) must first migrate into the peripheral tissues and then form immune synapses (IS) on target antigen-presenting cells. Both migration and IS formation involve mechanical force generation that is sustained by actin cytoskeletal rearrangement, a dynamic process of polymerizing G-actin monomers into F-actin filaments. Actin dynamics are important for generating structures such as the lamellipodia of migrating cells, the microvilli and mobile signalsomes at the IS, as well as synaptic pushing-and-pulling forces that have been implicated in maximizing cytolytic efficiency. However, little is known of the energy sources that fuel such mechanical activities. Actin dynamics are expected to be energetically demanding, given that actin is an ATPase and each G-actin hydrolyzes one ATP during assembly into filaments. In this study, we observed CTLs migrating and forming IS under various metabolic stresses. We discovered that during long-term migration, CTLs utilize nutrients in the environment, as real-time exchange of media into salt solutions within a perfusion chamber led to cells slowing down significantly. To measure force exertion at the IS, CTLs were treated with various inhibitors of metabolic pathways while forming synapses against deformable hydrogel balls. Using a custom computational pipeline, we found that synaptic forces depend on multiple metabolic pathways such as glycolysis and oxidative phosphorylation. Our study highlights a hitherto unexplored connection between cellular metabolism and mechanical activities integral to T-cell function and immune protection. This is of particular interest in the context of T-cell exhaustion, whereby CTLs chronically stimulated in metabolically inhospitable environments, such as tumors, develop into hypofunctional exhausted T cells. We suspect that immune hypo-functionality in these contexts may also involve defects in migration and IS formation due to the metabolic demand of these mechanical activities.

The intestinal microenvironment shapes macrophage and dendritic cell identity and function

The gut comprises the largest body interface with the environment and is continuously exposed to nutrients, food antigens, and commensal microbes, as well as to harmful pathogens. Subsets of both macrophages and dendritic cells (DCs) are present throughout the intestinal tract, where they primarily inhabit the gut-associate lymphoid tissue (GALT), such as Peyer's patches and isolated lymphoid follicles. In addition to their role in taking up and presenting antigens, macrophages and DCs possess extensive functional plasticity and these cells play complementary roles in maintaining immune homeostasis in the gut by preventing aberrant immune responses to harmless antigens and microbes and by promoting host defense against pathogens. The ability of macrophages and DCs to induce either inflammation or tolerance is partially lineage imprinted, but can also be dictated by their activation state, which in turn is determined by their specific microenvironment. These cells express several surface and intracellular receptors that detect danger signals, nutrients, and hormones, which can affect their activation state. DCs and macrophages play a fundamental role in regulating T cells and their effector functions. Thus, modulation of intestinal mucosa immunity by targeting antigen presenting cells can provide a promising approach for controlling pathological inflammation. In this review, we provide an overview on the characteristics, functions, and origins of intestinal macrophages and DCs, highlighting the intestinal microenvironmental factors that influence their functions during homeostasis. Unraveling the mechanisms by which macrophages and DCs regulate intestinal immunity will deepen our understanding on how the immune system integrates endogenous and exogenous signals in order to maintain the host's homeostasis.

Novel α-mannose-functionalized poly(β-amino ester) nanoparticles as mRNA vaccines with increased antigen presenting cell selectivity in the spleen.

mRNA vaccination has emerged as a prominent therapy for the future of medicine. Despite the colossal advance in this technology and worldwide efficacy proof (ca. COVID vaccines), mRNA carriers still lack cell/tissue specificity, leading to possible side effects, and reduced efficacy among others. Herein we make use of the ubiquitous affinity of antigen-presenting cells (APC)s for glycosides to achieve specific targeting. To achieve this goal, we designed a new generation of α-mannosyl functionalized oligopeptide-terminated poly(β-aminoester). Fine formulation of these polymers with mRNA resulted in nanoparticles decorated with surface-exposed α-mannoses with sizes around 180 nm and positive surface charge. Notably, these particles maintained their properties after freeze-drying and subsequent redispersion. Finally, our mRNA carriers preferentially targeted and transfected APCs in vitro and in vivo. In conclusion, we demonstrated, at a preclinical level, that the mannose functionalization enables more selective targeting of APCs and, thus, these polymer and nanoparticles are candidates for a new generation of mRNA immunotherapy vaccines.

Bridging nanoplatform and vaccine delivery, a landscape of strategy to enhance nasal immunity.

Vaccination is an urgently needed and effective option to address epidemic, cancers, allergies, and other diseases. Nasal administration of vaccines offers many benefits over needle-based injection including high compliance and less risk of infection. Inactivated or attenuated vaccines as convention vaccine present potential risks of pathogenic virulence reversal, the focus of nasal vaccine development has shifted to the use of next-generation (subunit and nucleic acid) vaccines. However, subunit and nucleic acid vaccine intranasally have numerous challenges in development and utilization due to mucociliary clearance, mucosal epithelial tight junction, and enzyme/pH degradation. Nanoplatforms as ideal delivery systems, with the ability to enhance the retention, penetration, and uptake of nasal mucosa, shows great potential in improving immunogenic efficacy of nasal vaccine. This review provides an overview of delivery strategies for overcoming nasal barrier, including mucosal adhesion, mucus penetration, targeting of antigen presenting cells (APCs), enhancement of paracellular transportation. We discuss methods of enhancing antigen immunogenicity by nanoplatforms as immune-modulators or multi-antigen co-delivery. Meanwhile, we describe the application status and development prospect of nanoplatforms for nasal vaccine administration. Development of nanoplatforms for vaccine delivery via nasal route will facilitate large-scale and faster global vaccination, helping to address the threat of epidemics.

33781 Sustained and durable response up to 24 weeks after last dose observed in a phase 2a study (NCT03754309) of amlitelimab (KY1005, SAR445229) a novel nondepleting anti-OX40Ligand (OX40L) mAb in patients with moderate to severe atopic dermatitis (AD)

Introduction: Amlitelimab (KY1005) is novel human nondepleting IgG4 monoclonal antibody blocking OX40Ligand which is found on antigen presenting cells (APCs) including dendritic cells. By targeting APCs it was hypothesised amlitelimab could potentially have long and durable anti-inflammatory effect in AD. Materials and methods: Kymab, a Sanofi Company, funded a Ph2a RCT (NCT03754309) in 89 moderate-to-severe AD patients (EASI>16, vIGA-AD>2 at baseline (BL)) with an inadequate response or intolerance of topical treatments. 1:1:1 randomization to intravenous amlitelimab low dose (LD, 200 mg loading/100 mg maintenance every 4 weeks), high dose (HD, 500 mg/250 mg Q4W) or placebo (PBO). Last dose was at Week 12. To assess long-term durability of response in patients achieving vIGA-AD0/1 at Week 16 efficacy assessments were collected to Week 36. Co-primary endpoints were % change in EASI from baseline and incidence of TEAEs at Week 16. Results: 88 patients received study medication. 59 evaluable patients competed Week 16. Both co-primary endpoints were met. 24 patients (12 LD (44%), 10 HD (37%) and 2 PBO (8%)) achieved a vIGA-AD 0/1 at Week 16. At Week 24, 83% (n = 10) LD, 90% (n = 9) HD, and 50% (n = 1) PBO maintained vIGA-AD0/1. 3 patients withdrew by Week 36 (2 LD, 1 HD). 21 patients remained with 67% (n = 8) LD, 70% (n = 7) HD, and 50% (n = 1) PBO maintaining a vIGA-AD0/1. Conclusions: This Ph2a study has demonstrated that Amlitelimab monotherapy targeting APCs is not only associated with a marked clinical improvement at Week16 but in patients achieving ‘clear’ or ‘almost clear’ skin (vIGA0/1) a sustained response up to 24 weeks after the last dose was maintained. A Phase 2b study is planned.

A Potent Micron Neoantigen Tumor Vaccine GP-Neoantigen Induces Robust Antitumor Activity in Multiple Tumor Models.

Therapeutic tumor neoantigen vaccines have been widely studied given their good safety profile and ability to avoid central thymic tolerance. However, targeting antigen‐presenting cells (APCs) and inducing robust neoantigen‐specific cellular immunity remain challenges. Here, a safe and broad‐spectrum neoantigen vaccine delivery system is proposed (GP‐Neoantigen) based on β‐1,3‐glucan particles (GPs) derived from Saccharomyces cerevisiae and coupling peptide antigens with GPs through convenient click chemistry. The prepared system has a highly uniform particle size and high APC targeting specificity. In mice, the vaccine system induced a robust specific CD8+ T cell immune response and humoral immune response against various conjugated peptide antigens and showed strong tumor growth inhibitory activity in EG7·OVA lymphoma, B16F10 melanoma, 4T1 breast cancer, and CT26 colon cancer models. The combination of the toll‐like receptors (TLRs) agonist PolyI:C and CpG 2395 further enhanced the antitumor response of the particle system, achieving complete tumor clearance in multiple mouse models and inducing long‐term rejection of reinoculated tumors. These results provide the broad possibility for its further clinical promotion and personalized vaccine treatment.

IMMU-18. Targeting Antigen Presenting Cells to improve virotherapy efficacy in Diffuse Midline Gliomas

With a 2-year survival less than 20%, Diffuse Intrinsic Pontine Glioma (DIPG) is the principal cause of pediatric death. Despite recent advances in the current treatments, the outcome for children with DIPGs remains dismal. Since the approval of T-VEC for melanoma by the FDA, oncolytic adenoviruses have emerged as a promising therapeutic strategy for brain tumors. Thus, our group launched the first world clinical trial phase I with the oncolytic adenovirus Delta-24-RGD (DNX-2401 in the clinic) for newly diagnosed DIPG (NCT03178032), which has shown safety and feasibility. Despite DNX-2401 increases the recruitment of T cells into the tumor, they usually become inactive due to the non-responsive tumor microenvironment evidencing the urgent need to improve this strategy focusing on the generation of effective long-term immune responses. Therefore, we decided to combine the Delta-24-RGD with the targeting of the costimulatory molecule CD40 in immunocompetent mice bearing orthotopic DIPG. The activation of the CD40 receptor, which is expressed by antigen presenting cells (APC) such as microglia, macrophages, and dendritic cells, is known to increase antigen presentation and enable T-cell priming and activation. Here, we observed that in addition to Delta-24-RGD anti-tumor effects, the stimulation of CD40 (using an agonistic antibody) on the tumor APCs results in a remodeling of the tumor immune compartment towards a proinflammatory scenario and a more efficient T-cell infiltration. Of importance, the combination therapy extends survival of treated mice as compared to single treatments or non-treated mice. In addition, we observe a complete regression of tumors in more than 40% of treated mice and the development of long-term anti-tumor immunity. We believe that these results provide a translational breakthrough in the treatment of these lethal tumors and open the door for a future innovative clinical trial.

Engineering mannosylated pickering emulsions for the targeted delivery of multicomponent vaccines

While research on cancer vaccines has made great strides in the field of immunotherapy, the targeted delivery of multiple effective components (rational-tailored antigens and adjuvants) remains a challenge. Here, we utilized the unique hierarchical structures of Pickering emulsions (particles, oil core, and water-oil interface) to develop mannosylated (M) Pickering emulsions (PE) that target antigen presenting cells and synergistically deliver antigenic peptides and the TLR9 agonist CpG (C) as an enhanced cancer vaccine (MPE-C). We chemically linked mannose residues to PLGA/PLAG-PEG nanoparticles and produced a dense array of mannose on the nanopatterned surface of Pickering emulsions, allowing for increased cellular targeting. Together with the inherent deformability of the oily core, MPE-C increased the droplet-cellular contact area and provoked the cellular recognition of mannose and CpG for enhanced immune activation. We found that MPE-C attracted a large number of APCs to the local site of administration, evidently increasing cellular uptake and activation. Additionally, we observed increased antigen-specific cellular immune responses, with potent anti-tumor effects against both E.G7-OVA and B16-MUCI tumors. Furthermore, MPE-C combined with PD-1 antibodies produced a significant tumor regression, resulting in synergistic increases in anti-tumor effects. Thus, through the strategic loading of mannose, antigens, and CpG, Pickering emulsions could serve as a targeted delivery platform for enhanced multicomponent cancer vaccines.

Targeting Antigen-Presenting Cells in Multiple Sclerosis Treatment

Multiple sclerosis (MS) is common neurological disease of the central nervous system (CNS) affecting mostly young adults. Despite decades of studies, its etiology and pathogenesis are not fully unraveled and treatment is still insufficient. The vast majority of studies suggest that the immune system plays a major role in MS development. This is also supported by the effectiveness of currently available MS treatments that target immunocompetent cells. In this review, the role of antigen-presenting cells (APC) in MS development as well as the novel therapeutic options targeting those cells in MS are presented. It is known that in MS, peripheral self-antigen-specific immune cells are activated during antigen presentation process and they enter the CNS through the disrupted blood–brain barrier (BBB). Myelin-reactive CD4+ T-cells can be activated by dendritic cells, infiltrating macrophages, microglia cells, or B-cells, which all express MHC class II molecules. There are also suggestions that brain endothelial cells may act as non-professional APCs and present myelin-specific antigens with MHC class II. Similarly, astrocytes, the major glial cells in the CNS, were shown to act as non-professional APCs presenting myelin antigens to autoreactive T-cells. Several currently available MS drugs such as natalizumab, fingolimod, alemtuzumab, and ocrelizumab may modulate antigen presentation in MS. Another way to use this mechanism in MS treatment may be the usage of specific tolerogenic dendritic cells or the induction of tolerance to myelin antigens by peptide vaccines.

Novel delivery mechanisms for antigen-specific immunotherapy.

Current therapies for autoimmune disorders often employ broad suppression of the immune system. Antigen-specific immunotherapy (ASI) seeks to overcome the side-effects of immunosuppressive therapy by specifically targeting only disease-related autoreactive T and B cells. Although it has been in development for several decades, ASI still is not in use clinically to treat autoimmunity. Novel ways to deliver antigen may be effective in inducing ASI. Here we review recent innovations in antigen delivery. New ways to deliver antigen include particle and nonparticle approaches. One main focus has been the targeting of antigen-presenting cells in a tolerogenic context. This technique often results in the induction and/or expansion of regulatory T cells, which has the potential to be effective against a complex, polyclonal immune response. Whether novel delivery approaches can help bring ASI into general clinical use for therapy of autoimmune diseases remains to be seen. However, preclinical work and early results from clinical trials using these new techniques show promising signs.

Abstract S05-02: Plant viral nanoparticle-based adjuvants for cancer immunotherapy and COVID-19 vaccines

Abstract Plant virus nanoparticles are recognized as a platform technology for potential applications in nanomedicine. While non-infectious toward mammals, many plant virus nanoparticles are recognized by the immune system and act as potent adjuvants, both in the classical setting of vaccination and cancer immunotherapy. In the context of cancer immunotherapy, we have demonstrated potent efficacy of plant virus, particularly cowpea mosaic virus (CPMV) when applied intratumorally; CPMV signals through and activates multiple toll-like receptors, which primes a potent innate immune activation leading to reprogramming of the tumor microenvironment from an immunosuppressed to immune-activated state. Data in mouse models of melanoma, colon cancer, ovarian cancer, glioma, and breast cancer demonstrate potent efficacy through priming of the innate immune system leading to adaptive and systemic anti-tumor immunity and most importantly immune memory. Efficacy was also replicated in canine patients with melanoma. Given the promise of the plant virus nanotechnology as an adjuvant for cancer immunotherapy, we also have begun to utilize the technology as a vaccination platform for the generation of COVID-19 vaccine candidates. We formulated multivalent COVID-19 vaccine candidates using plant virus nanoparticles displaying SARS-CoV-2 S protein peptide B cell epitopes. The plant virus nanoparticle confers efficient lymphatic trafficking and targeting of antigen-presenting cells; the adjuvant properties of the nanotechnology lead to robust antibody levels against the target, S protein. While other vaccine candidates have advanced through clinical testing, our approach has unique advantages: The high thermal and pH stability of the plant virus nanotechnology alleviates cold chain requirements; further, the stability of the platform technology enables its integration into vaccine delivery devices such as microneedle patches that can be shipped globally and be self-administered. In this presentation, we will discuss our data on the cancer immunotherapy and highlight the potential to pivot the technology for infectious disease vaccines with unique attributes. This work was funded in part through grants from the National Cancer Institute (U01CA218292) and National Science Foundation (RAPID CMMI-2027668). Citation Format: Nicole F. Steinmetz, Jonathan K. Pokorski, Steven N. Fiering, P. Jack Hoopes, Sourabh Shukla, Oscar A. Ortega-Rivera, Steven King, Cyril J. Empig. Plant viral nanoparticle-based adjuvants for cancer immunotherapy and COVID-19 vaccines [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr S05-02.

Preparation of mRNA Polyplexes with Post-conjugated Endosome-Disruptive Peptides.

Successful delivery of mRNA into the cytosol of professional antigen-presenting cells (APCs) poses one of the biggest challenges in developing effective mRNA vaccines to treat various cancers and viral infectious diseases. However, most polymeric mRNA delivery systems fail to transfect APCs. We have discovered that decoration of pH-sensitive endosome-disruptive GALA peptides on the surface of mRNA polyplexes leads to efficient targeting and transfection of APCs. GALA peptides not only enhance specific uptake in APCs through binding to sialic acid moieties, they also facilitate the endosomal escape of mRNA especially in dendritic cells (DCs). Here, we describe in detail the production of stabilized mRNA polyplexes post-conjugated with GALA peptides via copper-free click chemistry. Methods described here include the synthesis and purification of GALA peptides and its conjugation to mRNA polyplexes.