Activation Of Antigen-presenting Cells Research Articles

Abstract 5311: KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, stimulates anti-tumor immunity resulting in sustained anti-tumor effect against mouse intrahepatic tumor without hepatotoxicity

Abstract Several anti-CD40 agonist antibodies have been evaluated in clinical trials based on their preclinical antitumor activity via generation of antitumor innate and adaptive immunity through activation of antigen-presenting cells. However, conventional monospecific anti-CD40 agonist antibodies have faced limitations in terms of safety margins and clinical efficacy. To address this problem, we developed KK2269, a novel bispecific antibody (REGULGENT™ format) that binds to both CD40 and epithelial cell adhesion molecule (EpCAM), which is highly expressed in several types of tumors. KK2269 demonstrated EpCAM-dependent activation of CD40 signals both in vitro and in vivo, confirming that it is a tumor-targeting CD40 agonist. Liver metastasis is common in EpCAM-positive cancers, including colon, stomach, and lung cancers. Intrahepatic tumors can attenuate the efficacy of immune checkpoint inhibitors due to the liver tumor-specific suppressive immune milieu. Although CD40 agonists are expected to overcome the immunosuppressive environment, non-tumor-targeting CD40 agonists cause hepatotoxicity. In the present study, we used a syngeneic intrahepatic tumor model to examine whether KK2269 exhibits antitumor efficacy without causing hepatotoxicity. Firstly, we confirmed that KK2269 administration did not increase aspartate aminotransferase (AST) or cause pathological abnormalities in the liver despite Il12b induction in the EpCAM-positive tumor. Additionally, gene expression analysis of peritumoral normal liver revealed that KK2269 did not induce inflammatory cytokines. In contrast, a conventional anti-CD40 agonist antibody increased AST, caused pathological abnormalities, and upregulated inflammatory cytokines in peritumoral normal liver, as reported in the clinical setting. Secondly, the antitumor effect of KK2269 in combination with docetaxel was evaluated. The combination therapy synergistically reduced intrahepatic tumor volume, and the effect persisted even 21 days after the administration. Gene expression analysis revealed a lower immune infiltration in the intrahepatic tumor versus subcutaneous tumor and that KK2269 in combination with docetaxel increased T-cell infiltration and activation even in the immunosuppressive intrahepatic tumor. In summary, our study demonstrated that KK2269 activated dendritic cells in an EpCAM-positive tumor in the liver but did not affect peritumoral normal liver tissues. In addition, KK2269 in combination with docetaxel showed potent antitumor effect accompanied by T-cell infiltration and activation against the intrahepatic tumor, which has a lower immune infiltration. These results suggest that the combination therapy of KK2269 and docetaxel is promising for metastatic EpCAM-positive tumors. Citation Format: Yoshiki Sumitomo, Kyoko Kawasaki, Yuta Tezuka, Masato Saito, Akari Yao, Munetoshi Ando. KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, stimulates anti-tumor immunity resulting in sustained anti-tumor effect against mouse intrahepatic tumor without hepatotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5311.

DAMPening Tumor Immune Escape: The Role of Endoplasmic Reticulum Chaperones in Immunogenic Chemotherapy.

Significance: Preclinical and clinical research in the past two decades has redefined the mechanism of action of some chemotherapeutics that are able to activate the immune system against cancer when cell death is perceived by the immune cells. This immunogenic cell death (ICD) activates antigen-presenting cells (APCs) and T cells to induce immune-mediated tumor clearance. One of the key requirements to achieve this effect is the externalization of the damage-associated molecular patterns (DAMPs), molecules released or exposed by cancer cells during ICD that increase the visibility of the cancer cells by the immune system. Recent Advances: In this review, we focus on the role of calreticulin (CRT) and other endoplasmic reticulum (ER) chaperones, such as the heat-shock proteins (HSPs) and the protein disulfide isomerases (PDIs), as surface-exposed DAMPs. Once exposed on the cell membrane, these proteins shift their role from that of ER chaperone and regulator of Ca2+ and protein homeostasis to act as an immunogenic signal for APCs, driving dendritic cell (DC)-mediated phagocytosis and T-mediated antitumor response. Critical Issues: However, cancer cells exploit several mechanisms of resistance to immune attack, including subverting the exposure of ER chaperones on their surface to avoid immune recognition. Future Directions: Overcoming these mechanisms of resistance represents a potential therapeutic opportunity to improve cancer treatment effectiveness and patient outcomes.

Abstract 5572: Microneedle-guided lymphatic delivery of a natural immune modulator toxin for enhanced cancer immunotherapy

Abstract Cancer immunotherapy has demonstrated significant potential as a cancer treatment by enhancing the immune system's ability to recognize and eliminate cancer cells. However, its efficacy can be limited by factors such as tumor heterogeneity, immunosuppressive tumor microenvironments, and systemic toxicities. Recent advances in drug delivery systems have facilitated the development of more targeted and personalized cancer therapies. Microneedles have emerged as a promising platform for non-invasive and highly localized drug delivery, directly administering drugs, vaccines, and other therapeutic agents to the skin. In this study, we designed dissolving microneedles (dMN) based on a biocompatible amphiphilic tri-block copolymer, which enables the self-assembly of nano-micelles containing hydrophobic drugs when applied to the skin. We used the dMN technology to formulate SKKU-06, a hydrophobic natural immune modulator toxin derived from fungi, which exhibits anti-cancer and immunomodulatory properties in melanoma (SKKU-06@dMN). After intratumoral application of SSKU-06@dMN to skin tumors, the drug-loaded nano-micelles can migrate to tumor-draining lymph nodes (TDLN). The dMN-guided delivery of SKKU-06 to skin tumors and TDLN induced immunogenic cell death and stimulated the activation and maturation of antigen-presenting cells (APCs), promoting the development of humoral and cellular anti-tumor immunity. Furthermore, the immunomodulatory effects of SSKU-06@dMN were enhanced when combined with anti-PD-1 treatment, impacting the tumor microenvironment through increased intratumoral CD8+ T cell infiltration and reduced Treg populations. This resulted in efficient growth inhibition of established skin cancer and metastatic cancer, as well as prolonged survival. The dMN-guided lymphatic delivery of SKKU-06 demonstrates the potential for treating metastatic solid tumors and improving cancer immunotherapy efficacy by modulating the tumor microenvironment. Citation Format: Su Yeon Lim, Byung Deok Kim, PIN LIU, Ju hwa Shin, Ji Hoon Jeong. Microneedle-guided lymphatic delivery of a natural immune modulator toxin for enhanced cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5572.

Abstract 4103: Novel mRNA-encoded HPV vaccine with APC maturation signal and endolysosomal trafficking domain demonstrates superior T cell-mediated immunogenicity and efficacious tumor clearance in vivo

Abstract Human Papillomavirus (HPV), particularly HPV16, is a known causative agent of cancers in various anatomical sites, notably the anogenital and oropharyngeal regions. Despite the availability of prophylactic HPV vaccines for almost two decades, vaccination rates among young females, who are at a heightened risk for cervical intraepithelial neoplasia (CIN) and subsequent invasive cancer, remain suboptimal. The current standard of care for cervical cancer, involving invasive surgery and chemotherapy-based treatments, underscores the pressing need for safer and more effective therapeutics to enhance patient survival and quality of life. In this context, while several HPV16-E6/E7 targeted therapeutic vaccines are in development and clinical trials, their clinical efficacy has been limited due to inadequate antigen presentation and suboptimal immunogenicity. Addressing this, our team has developed a novel, highly immunogenic mRNA therapeutic cancer vaccine, engineered to activate a robust T cell-mediated adaptive immune response and exhibit strong anti-tumor potential. Our innovative approach includes the design of an HPV16-E6/E7 mRNA vaccine (ABO2101) that incorporates a unique antigen-expressing cassette. This cassette features endolysosomal trafficking domains and antigen presenting cell (APC) activation stimulators, enhancing both Class I and Class II antigen presentation and APC maturation, thereby amplifying antigen-specific T cell responses. In both murine and human-derived experimental models, ABO2101 has demonstrated a significantly more potent adaptive T cell response against HPV16 antigens compared to other HPV16 mRNA therapeutic vaccines. In antitumor efficacy evaluations using TC-1 tumor-bearing mice, ABO2101 single-dose administration successfully eradicated both early-stage (50 mm³) and advanced (500 mm³) tumors. This was accompanied by a marked increase in HPV16-specific CD8+ cytotoxic T lymphocytes within the tumor microenvironment. Moreover, in prophylactic and adjuvant therapy models, ABO2101 prevented tumor growth across various dosages, indicating its robust preventative capabilities. Further, the combination of ABO2101 with immune checkpoint inhibitors, including PD-1 and CTLA-4 antibodies, displayed a synergistic effect in both immune-oncology sensitive and resistant animal models, suggesting a potential for combinational clinical therapy. In conclusion, our development of ABO2101, a mRNA-based HPV16-E6/E7 therapeutic cancer vaccine inclusive of APC maturation signals, represents a groundbreaking advancement in HPV-associated cancer treatment, as supported by our compelling preclinical data. Citation Format: Liang Du, Hongya Han, Jingshu Ma, Zhenxing Yang, Jinjuan Mao, Jijun Yuan, Bo Ying. Novel mRNA-encoded HPV vaccine with APC maturation signal and endolysosomal trafficking domain demonstrates superior T cell-mediated immunogenicity and efficacious tumor clearance in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4103.

Abstract 4043: Nanoparticle-mediated combination therapy to synergistically harness Type I interferons and senescence in the pancreatic tumor microenvironment

Abstract We seek to combine a unique innate immunomodulatory nanoparticle (NP) system with RAS inhibitors for a multi-faceted therapy that mitigates the formidable drug delivery challenges in pancreatic ductal adenocarcinoma (PDAC). PDAC has quickly risen to the third most deadly cancer largely due to its fibrotic, desmoplastic, and immunosuppressive tumor microenvironment (TME) that limits delivery and efficacy of chemo- and immunotherapeutics. We have engineered unique lipid-based NPs that co-encapsulate agonists of the Stimulator of Interferon Genes (STING) and Toll-like Receptor 4 (TLR4) pathways. NPs can be safely delivered in the systemic blood circulation to achieve TME deposition, uptake by innate antigen-presenting cells (APCs), and robust, synergistic production of Type I interferons by dual STING/TLR4 activation. Here, we hypothesize that combination therapy of proinflammatory NPs with tumor senescence-inducing RAS inhibitors, tremetinib and palbociclib, will not only augment CD8+ T cell recruitment to the TME but enhance their sustained activation by mitigating local immunosuppression. In mice bearing orthotopic transplant KPC tumors or KPC GEMMs, combination NP/inhibitor treatment promoted significant NP delivery to tumors, APC and natural killer (NK) cell activation, and CD8+ T cell-mediated clearance, compared to monotherapies and untreated controls. Unexpectedly and strikingly, these studies also showed that, besides innate immune cells, combination therapy also promoted Type I interferon and other proinflammatory cytokine production by PDAC tumor cells, which are otherwise notoriously unresponsive to current state-of-the-art treatments. Further, in KPC GEMMs, 25% of mice exhibited apparent complete responses following combination treatment, which, to our knowledge, is challenging to achieve in this model. Current studies include the mechanistic elucidation of immune- and tumor-intrinsic factors that govern efficacy of our combination therapy. In conclusion, these findings strongly corroborate the use of this NP-based system as a platform therapy for similar drugs and across other aggressive cancers. They also strongly make the case for the rational design of combination therapies to achieve synergistic therapeutic outcomes with minimal systemic toxicities. Citation Format: Prabhani Atukorale, Marcus Ruscetti, Loretah Chibaya, Christina Lusi, Kelly DeMarco, Griffin Kane, Meghan Brassil, Chaitanya Parikh, Katherine Murphy. Nanoparticle-mediated combination therapy to synergistically harness Type I interferons and senescence in the pancreatic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4043.

Abstract 5309: ATOR-4066, a Neo-X-Prime™ bispecific antibody targeting CD40 and CEACAM5, induces tumor localized immune cell activation in preclinical in vivo tumor model

Abstract ATOR-4066 is a preclinical stage bispecific antibody targeting CD40 and CEACAM5, based on Alligator’s novel Neo-X-Prime™ antibody technology platform. ATOR-4066 induces a neoantigen specific T-cell response by simultaneous binding to CD40 on myeloid cells such as antigen presenting cells (APCs) and to CEACAM5 on tumor cells. CEACAM5 is a tumor-associated antigen highly expressed on several cancer indications such as colorectal and gastric cancer, as well as on tumor-derived material. ATOR-4066 treatment results in (1) CEACAM5-conditional CD40-mediated activation of APCs and (2) enhanced uptake of tumor-derived material, leading to cross-presentation of neoantigen and priming of neoantigen-specific T cells, resulting in enhanced tumor cell killing. We have previously shown evidence of a potent anti-tumor effect of ATOR-4066 in vivo, superior to the effects observed with the corresponding CD40 mAb. Moreover, we have demonstrated CEACAM5-dependent activation of CD40-expressing cells in vitro, and an ability to mediate co-localization of CEACAM5-expressing tumor debris and CD40 expressing APCs. In addition, ATOR-4066 was shown to induce CEACAM5-conditional activation of tumor-infiltrating CD40-expressing immune cells in dissociated primary human tumor material from colorectal and gastric cancer patients. Here we present data further elucidating the mode of action of ATOR-4066 in vivo. Following ATOR-4066 treatment, tumors and lymph nodes were isolated from human CD40 transgenic mice bearing MC38 tumors expressing human CEACAM5. Flow cytometry analysis shows an increased number of tumor-infiltrating immune cells in mice treated with ATOR-4066 compared to treatments with CD40 mAb or vehicle. The study also reveals upregulation of co-stimulatory molecules such as CD86 on DCs and macrophages after treatment with ATOR-4066 compared to groups treated with CD40 mAb or vehicle. The effect was localized to the tumor microenvironment and not observed in tumor draining lymph nodes, confirming previous in vitro results demonstrating CEACAM5-conditional immune activation. Moreover, RNA-sequencing of tumor samples displayed distinct transcriptomic alterations following ATOR-4066 treatment vs. CD40 mAb or vehicle treatment in vivo. In contrast, RNA-sequencing of peripheral blood in ATOR-4066 treated mice showed only small transcriptomic differences compared to the vehicle treated group, but distinct from mice treated with a CD40 mAb, further demonstrating the CEACAM5 dependent response of ATOR-4066. In summary, these preclinical data demonstrate that ATOR-4066 activates infiltrating myeloid cells resulting in increases in activated tumor infiltrating T cells, thus creating a pro-inflammatory tumor microenvironment, which strongly supports further development and clinical testing of ATOR-4066. Citation Format: Hampus Andersson, Ida Uddbäck, Mona Celander, Maria Johansson, Lill Ljung, Anneli Nilsson, Amulya Krishna Shetty, David Gomez Jimenez, Mattias Levin, Sara Fritzell, Laura von Schantz, Malin Lindstedt, Karin Hägerbrand, Anette Sundstedt, Peter Ellmark. ATOR-4066, a Neo-X-Prime™ bispecific antibody targeting CD40 and CEACAM5, induces tumor localized immune cell activation in preclinical in vivo tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5309.

Abstract 499: Enhancing neoantigen cancer vaccine efficacy with a self-assembling peptide conjugates nanoparticle platform

Abstract Tumor neoantigens, arising from genomic mutations during cancer cell proliferation, are absent in normal tissues, making them ideal immunotherapy targets. Predicting neoantigen peptide sequences using artificial neural networks (ANNs) like NetMHCpan offers a promising approach to developing personalized peptide cancer vaccines. However, limitations such as low affinity, short half-life, and weak immunogenicity hinder their efficacy. To address these challenges, we propose loading neoantigens into nanoparticles for protection and slow release within the body. We used ANN prediction to determine neoantigen peptide sequences for various cancer cell lines and engineered these peptides by conjugating them with Toll-like receptor 7 agonist (TLR7a) adjuvants, forming a self-assembling peptide conjugates nanoparticle (SPCN) platform. To assess the immunogenicity of the SPCN platform, we conducted enzyme-linked ELISpot assays and evaluated the activation of antigen-presenting cells (APCs) in draining lymph nodes using flow cytometry analysis. The therapeutic efficacy of the SPCN platform was determined by monitoring tumor growth curves in mouse models. The SPCN platform demonstrated broad applicability, carrying different neoantigens in vivo, resulting in robust and safe systemic anti-tumor responses. The SPCN enhanced immunogenicity, stimulating splenocytes to produce more interferon gamma and facilitating efficient absorption by antigen-presenting cells (APCs). Moreover, SPCNs loaded with neoantigens targeted different cancer models, including B16OVA, MB49, and RIL175. In conclusion, our self-assembling peptide conjugates nanoparticle platform enhances APC activation and neoantigen immunogenicity, leading to improved anti-tumor efficacy, offering valuable insights for developing synthetic peptide cancer vaccines. Citation Format: Yangfan Wu, Jian-Dong Huang. Enhancing neoantigen cancer vaccine efficacy with a self-assembling peptide conjugates nanoparticle platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 499.

Abstract 288: Middle-aged murine models of triple negative breast cancer (TNBC) show significant differences in tumor immune and stromal environments when compared to classic 6-8 week old models; Challenges and opportunities for preclinical efficacy studies

Abstract The immune system orchestra suffer a deficit of function during aging, and the incidence of the large majority of cancer types is significantly increased in elderly humans. Although BC can occur in young women, it is way more common in middle-age and elderly women. There is evidence that a tonic immune system is pivotal for the success of therapies aimed at awakening a dormant or exhausted immunological response against cancer cells by mean of checkpoint inhibitors (CI). However, rodent models of cancers are currently using very young mice of a few weeks of age, which have a fully functional and tonic immune system and thymus and thus might be poorly representative of adult human cancer patients. We have recently reported that that a triple therapy (TT) involving antigen-presenting cell activation by vinca alkaloids and the generation of new TCF1+ stem cell-like T cell (scT) clones by an alkylating agent can significantly improve the efficacy of the CI anti-PD-1 in models of cancer known to be otherwise CI-resistant such as TNBC and lymphoma (Falvo et al, Cancer Res 2021; Orecchioni et al, JCM 2023). TT effect was due to T cells, as it was abrogated by their in vivo depletion. In the present study we investigated differences in TNBC growth kinetics, TT preclinical activity and tumor microenvironment (TME, including intratumoral immune and stroma cells) in young (6-8w, representative of a 6y old human) versus adult (12m, representative of 40y humans) mice. Models included 4T1 and EMT6 TNBC cells, the former generating a mostly lymphoid TME and the latter generating a mostly myeloid TME. In both models, TT efficacy was similar in young and in adult mice, as the treatment abrogated TNBC local and metastatic growth. CD8+ (but not CD4+) scTs, likely crucial players in TT efficacy, were only slightly reduced in adult mice in spite of age-related thymus involution. This notwithstanding, single cell RNA (10x), IHC and flow cytometry analyses of immune (CD45+EPCAM-) and stroma (CD45-EPCAM-) cell populations indicated major differences in the TME of young vs adult mice. Adults had significantly less CD4+ scTs, B naïve and NK cells and significantly increased memory B cells. TME matrix CAF were skewed in young, while pro-inflammatory stromal populations and myofibroblasts were skewed in adults. Matrix CAFs down regulated a signature involved in different ECM-remodeling abilities, and up regulated metabolic and hypoxic pathways. Expression of genes encoding for glycoproteins, basement membrane components, and collagens were also upregulated. Our data indicate profound differences between young and adult mice immune and stromal TMEs that should be taken into consideration when selecting the age of mice to be used in preclinical efficacy studies in orthotopic immune competent mice. Citation Format: Paolo Falvo, Stephan Gruener, Stefania Orecchioni, Giovanna Talarico, Giulia Bravetti, Giulia Mitola, Federica Pisati, Iros Barozzi, Francesco Bertolini. Middle-aged murine models of triple negative breast cancer (TNBC) show significant differences in tumor immune and stromal environments when compared to classic 6-8 week old models; Challenges and opportunities for preclinical efficacy studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 288.

A self-assembled, genetically engineered, irradiated tumor cell debris vaccine.

Vaccine-based therapeutics for cancers face several challenges including lack of immunogenicity and tumor escape pathways for single antigen targets. It has been reported that radiotherapy has an in situ vaccine effect that provides tumor antigens following irradiation, helping to activate antigen-presenting cells (APCs). Herein, a new vaccine approach is developed by combining genetically engineered irradiated tumor cell debris (RTD) and hyaluronic acid (HA), termed HA@RTD. A cancer cell line is developed that overexpresses granulocyte-macrophage colony-stimulating factor (GM-CSF). A hydrogel was developed by covalent conjugation of HA with RTD proteins that acted as a potent vaccine system, the effects which were probed with T cell receptor sequencing. The engineered vaccine activated antitumor immunity responses and prevented tumor growth in mice even with a single immunization. HA@RTD vaccine efficacy was also assessed in therapeutic settings with established tumors and in combination with immune checkpoint blockade.

Astragalus root increases Treg and Th17 involvement in embryo implantation and pregnancy maintenance by decreasing CTLA-4+ Tregs.

Maintenance of pregnancy is highly dependent on the maternal immune system. High levels of regulatory T cells (Tregs) accumulate in the maternal placenta to suppress immunoreactivity against fetal antigens. We assessed whether Astragalus root (AsR) and AsR-containing Kampo medicines modulate immunoreactivity and thereby increase mouse litter size. AsR-exposed murine splenocytes exhibited significantly increased IL-2 secretion. In AsR-exposed mice, total Tregs were significantly increased, whereas cytotoxic T lymphocyte antigen 4 (CTLA-4)-positive Tregs were decreased in AsR-exposed mice. Tregs express IL-2 receptor subunit alpha and are activated by IL-2. CTLA-4 interacts with B7 expressed in antigen-presenting cells (APCs) with high affinity, and CTLA-4/B7 signaling plays a critical role in inhibiting APC activity, thereby suppressing CD4+ T cell proliferation and activation. The decrease in CTLA-4+ Tregs in AsR-exposed mice is thought to induce an increase in CD4+ T cells, leading to increased IL-2 secretion from CD4+ T cells followed by Treg activation. Th17 cells prevent trophoblast apoptosis, resulting in trophoblast invasion into the decidua. AsR increases Th17 cells, thereby inducing dose-dependent increases in litter size. Although Keishikaogito (KO)- and Ogikenchuto (OK)-exposed mice exhibited increased IL-2 secretion and splenic Tregs, KO also increased CTLA-4+ Tregs. Therefore, KO promoted immunosuppression by increasing CTLA-4+ Tregs, which induced a decrease in Th17 and exerted little effect on litter size. Therefore, an increase in both Tregs and Th17 cells can be considered necessary for embryo implantation and pregnancy maintenance.

Anti-cathepsin D immunotherapy triggers both innate and adaptive anti-tumour immunity in breast cancer.

Triple-negative breast cancer (TNBC) has poorer outcomes than other breast cancers (BC), including HER2+ BC. Cathepsin D (CathD) is a poor prognosis marker overproduced by BC cells, hypersecreted in the tumour microenvironment with tumour-promoting activity. Here, we characterized the immunomodulatory activity of the anti-CathD antibody F1 and its improved Fab-aglycosylated version (F1M1) in immunocompetent mouse models of TNBC (C57BL/6 mice harbouring E0771 cell grafts) and HER2-amplified BC (BALB/c mice harbouring TUBO cell grafts). CathD expression was evaluated by western blotting and immunofluorescence, and antibody binding to CathD by ELISA. Antibody anti-tumour efficacy was investigated in mouse models. Immune cell recruitment and activation were assessed by immunohistochemistry, immunophenotyping, and RT-qPCR. F1 and F1M1 antibodies remodelled the tumour immune landscape. Both antibodies promoted innate antitumour immunity by preventing the recruitment of immunosuppressive M2-polarized tumour-associated macrophages (TAMs) and by activating natural killer cells in the tumour microenvironment of both models. This translated into a reduction of T-cell exhaustion markers in the tumour microenvironment that could be locally supported by enhanced activation of anti-tumour antigen-presenting cell (M1-polarized TAMs and cDC1 cells) functions. Both antibodies inhibited tumour growth in the highly-immunogenic E0771 model, but only marginally in the immune-excluded TUBO model, indicating that anti-CathD immunotherapy is more relevant for BC with a high immune cell infiltrate, as often observed in TNBC. Anti-CathD antibody-based therapy triggers the anti-tumour innate and adaptive immunity in preclinical models of BC and is a promising immunotherapy for immunogenic TNBC.

Activation of RIG-I/MDA5 Signaling and Inhibition of CD47-SIRPα Checkpoint with a Dual siRNA-Assembled Nanoadjuvant for Robust Cancer Immunotherapy.

Antigen-presenting cells (APCs) play a crucial role in the anti-tumor immunity as they are responsible for capturing, processing, and presenting tumor antigens to T cells. However, their activation is often limited by the absence of adjuvants and the suppressive effects of immune checkpoints, such as CD47-SIRPα. Herein, we present a nanoadjuvant that is self-assembled from long RNA building blocks generated through rolling circle transcription (RCT) reaction and further modified with cationic liposomes. Owing to the high load of densely packed RNA, this nanoadjuvant could robustly activate RIG-I/MDA5 signaling in APCs, leading to the maturation of dendritic cells (DCs) and the polarization of tumor-associated macrophages (TAMs) toward an anti-tumor M1-like phenotype. In addition, with a well-designed template, the generated long RNA from RCT reaction includes two kinds of siRNA targeting both CD47 in tumor cells and SIRPα in APCs. This dual gene silencing results in efficient inhibition of the CD47-SIRPα checkpoint. Collectively, the robust activation of RIG-I/MDA5 signaling and efficient inhibition of CD47-SIRPα checkpoint enhance the phagocytic activity of APCs, which in turn promotes the cross-priming of effector T cells and the activation of anti-tumor immune responses. This study therefore provides a simple and robust RNA nanoadjuvant for cancer immunotherapy.

Activation of RIG‐I/MDA5 Signaling and Inhibition of CD47‐SIRPα Checkpoint with a Dual siRNA‐Assembled Nanoadjuvant for Robust Cancer Immunotherapy

AbstractAntigen‐presenting cells (APCs) play a crucial role in the anti‐tumor immunity as they are responsible for capturing, processing, and presenting tumor antigens to T cells. However, their activation is often limited by the absence of adjuvants and the suppressive effects of immune checkpoints, such as CD47‐SIRPα. Herein, we present a nanoadjuvant that is self‐assembled from long RNA building blocks generated through rolling circle transcription (RCT) reaction and further modified with cationic liposomes. Owing to the high load of densely packed RNA, this nanoadjuvant could robustly activate RIG‐I/MDA5 signaling in APCs, leading to the maturation of dendritic cells (DCs) and the polarization of tumor‐associated macrophages (TAMs) toward an anti‐tumor M1‐like phenotype. In addition, with a well‐designed template, the generated long RNA from RCT reaction includes two kinds of siRNA targeting both CD47 in tumor cells and SIRPα in APCs. This dual gene silencing results in efficient inhibition of the CD47‐SIRPα checkpoint. Collectively, the robust activation of RIG‐I/MDA5 signaling and efficient inhibition of CD47‐SIRPα checkpoint enhance the phagocytic activity of APCs, which in turn promotes the cross‐priming of effector T cells and the activation of anti‐tumor immune responses. This study therefore provides a simple and robust RNA nanoadjuvant for cancer immunotherapy.

Human In vitro Modeling Identifies Adjuvant Combinations that Unlock Antigen Cross-presentation and Promote T-helper 1 Development in Newborns, Adults and Elders

Adjuvants are vaccine components that can boost the type, magnitude, breadth, and durability of an immune response. We have previously demonstrated that certain adjuvant combinations can act synergistically to enhance and shape immunogenicity including promotion of Th1 and cytotoxic T-cell development. These combinations also promoted protective immunity in vulnerable populations such as newborns. In this study, we employed combined antigen-specific human in vitro models to identify adjuvant combinations that could synergistically promote the expansion of vaccine-specific CD4+ cells, induce cross-presentation on MHC class I, resulting in antigen-specific activation of CD8+ cells, and direct the balance of immune response to favor the production of Th1-promoting cytokines. A screen of 78 adjuvant combinations identified several T cell-potentiating adjuvant combinations. Remarkably, a combination of TLR9 and STING agonists (CpG + 2,3-cGAMP) promoted influenza-specific CD4+ and CD8+ T cell activation and selectively favored production of Th1-polarizing cytokines TNF and IL-12p70 over co-regulated cytokines IL-6 and IL-12p40, respectively. Phenotypic reprogramming towards cDC1-type dendritic cells by CpG + 2,3-cGAMP was also observed. Finally, we characterized the molecular mechanism of this adjuvant combination including the ability of 2,3-cGAMP to enhance DC expression of TLR9 and the dependency of antigen-presenting cell activation on the Sec22b protein important to endoplasmic reticulum-Golgi vesicle trafficking. The identification of the adjuvant combination CpG + 2,3-cGAMP may therefore prove key to the future development of vaccines against respiratory viral infections tailored for the functionally distinct immune systems of vulnerable populations such as older adults and newborns.

Synergistic Effect of Retinoic Acid and Lactoferrin in the Maintenance of Gut Homeostasis.

Lactoferrin (LF) is a glycoprotein that binds to iron ions (Fe2+) and other metallic ions, such as Mg2+, Zn2+, and Cu2+, and has antibacterial and immunomodulatory properties. The antibacterial properties of LF are due to its ability to sequester iron. The immunomodulatory capability of LF promotes homeostasis in the enteric environment, acting directly on the beneficial microbiota. LF can modulate antigen-presenting cell (APC) biology, including migration and cell activation. Nonetheless, some gut microbiota strains produce toxic metabolites, and APCs are responsible for initiating the process that inhibits the inflammatory response against them. Thus, eliminating harmful strains lowers the risk of inducing chronic inflammation, and consequently, metabolic disease, which can progress to type 2 diabetes mellitus (T2DM). LF and retinoic acid (RA) exhibit immunomodulatory properties such as decreasing cytokine production, thus modifying the inflammatory response. Their activities have been observed both in vitro and in vivo. The combined, simultaneous effect of these molecules has not been studied; however, the synergistic effect of LF and RA may be employed for enhancing the secretion of humoral factors, such as IgA. We speculate that the combination of LF and RA could be a potential prophylactic alternative for the treatment of metabolic dysregulations such as T2DM. The present review focuses on the importance of a healthy diet for a balanced gut and describes how probiotics and prebiotics with immunomodulatory activity as well as inductors of differentiation and cell proliferation could be acquired directly from the diet or indirectly through the oral administration of formulations aimed to maintain gut health or restore a eubiotic state in an intestinal environment that has been dysregulated by external factors such as stress and a high-fat diet.

Advancing nanotechnology for neoantigen-based cancer theranostics.

Neoantigens play a pivotal role in the field of tumour therapy, encompassing the stimulation of anti-tumour immune response and the enhancement of tumour targeting capability. Nonetheless, numerous factors directly influence the effectiveness of neoantigens in bolstering anti-tumour immune responses, including neoantigen quantity and specificity, uptake rates by antigen-presenting cells (APCs), residence duration within the tumour microenvironment (TME), and their ability to facilitate the maturation of APCs for immune response activation. Nanotechnology assumes a significant role in several aspects, including facilitating neoantigen release, promoting neoantigen delivery to antigen-presenting cells, augmenting neoantigen uptake by dendritic cells, shielding neoantigens from protease degradation, and optimizing interactions between neoantigens and the immune system. Consequently, the development of nanotechnology synergistically enhances the efficacy of neoantigens in cancer theranostics. In this review, we provide an overview of neoantigen sources, the mechanisms of neoantigen-induced immune responses, and the evolution of precision neoantigen-based nanomedicine. This encompasses various therapeutic modalities, such as neoantigen-based immunotherapy, phototherapy, radiotherapy, chemotherapy, chemodynamic therapy, and other strategies tailored to augment precision in cancer therapeutics. We also discuss the current challenges and prospects in the application of neoantigen-based precision nanomedicine, aiming to expedite its clinical translation.

Marked enhancement of the immunogenicity of plant-expressed IgG-Fc fusion proteins by inclusion of cholera toxin non-toxic B subunit within the single polypeptide.

Immunoglobulin G (IgG)-based fusion proteins have been widely exploited as a potential vaccine delivery platform but in the absence of exogenous adjuvants, the lack of robust immunity remains an obstacle. Here, we report on a key modification that overcomes that obstacle. Thus, we constructed an IgG-Fc vaccine platform for dengue, termed D-PCF, which in addition to a dengue antigen incorporates the cholera toxin non-toxic B subunit (CTB) as a molecular adjuvant, with all three proteins expressed as a single polypeptide. Following expression in Nicotiana benthamiana plants, the D-PCF assembled as polymeric structures of similar size to human IgM, a process driven by the pentamerization of CTB. A marked improvement of functional properties in vitro and immunogenicity in vivo over a previous iteration of the Fc-fusion protein without CTB [1] was demonstrated. These include enhanced antigen presenting cell binding, internalization and activation, complement activation, epithelial cell interactions and ganglioside binding, as well as more efficient polymerization within the expression host. Following immunization of mice with D-PCF by a combination of systemic and mucosal (intranasal) routes, we observed robust systemic and mucosal immune responses, as well as systemic T cell responses, significantly higher than those induced by a related Fc-fusion protein but without CTB. The induced antibodies could bind to the domain III of the dengue virus envelope protein from all four dengue serotypes. Finally, we also demonstrated feasibility of aerosolization of D-PCF as a prerequisite for vaccine delivery by the respiratory route.

The adjuvant BcfA activates antigen presenting cells through TLR4 and supports TFH and TH1 while attenuating TH2 gene programming.

Adjuvants added to subunit vaccines augment antigen-specific immune responses. One mechanism of adjuvant action is activation of pattern recognition receptors (PRRs) on innate immune cells. Bordetella colonization factor A (BcfA); an outer membrane protein with adjuvant function, activates TH1/TH17-polarized immune responses to protein antigens from Bordetella pertussis and SARS CoV-2. Unlike other adjuvants, BcfA does not elicit a TH2 response. To understand the mechanism of BcfA-driven TH1/TH17 vs. TH2 activation, we screened PRRs to identify pathways activated by BcfA. We then tested the role of this receptor in the BcfA-mediated activation of bone marrow-derived dendritic cells (BMDCs) using mice with germline deletion of TLR4 to quantify upregulation of costimulatory molecule expression and cytokine production in vitro and in vivo. Activity was also tested on human PBMCs. PRR screening showed that BcfA activates antigen presenting cells through murine TLR4. BcfA-treated WT BMDCs upregulated expression of the costimulatory molecules CD40, CD80, and CD86 and produced IL-6, IL-12/23 p40, and TNF-α while TLR4 KO BMDCs were not activated. Furthermore, human PBMCs stimulated with BcfA produced IL-6. BcfA-stimulated murine BMDCs also exhibited increased uptake of the antigen DQ-OVA, supporting a role for BcfA in improving antigen presentation to T cells. BcfA further activated APCs in murine lungs. Using an in vitro TH cell polarization system, we found that BcfA-stimulated BMDC supernatant supported TFH and TH1 while suppressing TH2 gene programming. Overall, these data provide mechanistic understanding of how this novel adjuvant activates immune responses.

Recent advances in peptide-based nanovaccines for re-emerging and emerging infectious diseases

Peptide-based nanovaccines have emerged as promising strategies for combating re-emerging and emerging infectious diseases. They exhibit excellent immunogenicity and therapeutic potential. They have shown the ability to elicit robust immune responses, including activation of antigen-presenting cells, induction of specific antibodies and T-cell responses, and generation of memory immune cells. This comprehensive review article aims to provide a thorough overview of recent advances in the field, including self-assembling peptide-based nanovaccines, immunological mechanisms, structural design approaches, and utilization of various nanomaterials. Overall, peptide-based nanovaccines hold great promise in combating infectious diseases. Precise design and assembly of targeted and tailored immune responses will enable effective prevention, treatment, and long-term protection. Further research is needed to optimize their efficacy, safety, and clinical translation. The knowledge gained from these studies will pave the way for a future with more effective immunotherapeutic interventions against infectious diseases.

The glycoimmune checkpoint receptor Siglec-7 interacts with T-cell ligands and regulates T-cell activation

Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is a glycan-binding immune receptor that is emerging as a significant target of interest for cancer immunotherapy. The physiological ligands that bind Siglec-7, however, remain incompletely defined. In this study, we characterized the expression of Siglec-7 ligands on peripheral immune cell subsets and assessed whether Siglec-7 functionally regulates interactions between immune cells. We found that disialyl core 1 O-glycans are the major immune ligands for Siglec-7 and that these ligands are particularly highly expressed on naïve T-cells. Densely glycosylated sialomucins are the primary carriers of these glycans, in particular a glycoform of the cell-surface marker CD43. Biosynthesis of Siglec-7-binding glycans is dynamically controlled on different immune cell subsets through a genetic circuit involving the glycosyltransferase GCNT1. Siglec-7 blockade was found to increase activation of both primary T-cells and antigen-presenting dendritic cells invitro, indicating that Siglec-7 binds T-cell glycans to regulate intraimmune signaling. Finally, we present evidence that Siglec-7 directly activates signaling pathways in T-cells, suggesting a new biological function for this receptor. These studies conclusively demonstrate the existence of a novel Siglec-7-mediated signaling axis that physiologically regulates T-cell activity. Going forward, our findings have significant implications for the design and implementation of therapies targeting immunoregulatory Siglec receptors.