Immune Adjuvant Research Articles

The Papain-like Protease Domain of Severe Acute Respiratory Syndrome Coronavirus 2 Conjugated with Human Beta-Defensin 2 and Co1 Induces Mucosal and Systemic Immune Responses against the Virus.

Most of the licensed vaccines against SARS-CoV-2 target spike proteins to induce viral neutralizing antibodies. However, currently prevalent SARS-CoV-2 variants contain many mutations, especially in their spike proteins. The development of vaccine antigens with conserved sequences that cross-react with variants of SARS-CoV-2 is needed to effectively defend against SARS-CoV-2 infection. Given that viral infection is initiated in the respiratory mucosa, strengthening the mucosal immune response would provide effective protection. We constructed a mucosal vaccine antigen using the papain-like protease (PLpro) domain of non-structural protein 3 of SARS-CoV-2. To potentiate the mucosal immune response, PLpro was combined with human beta-defensin 2, an antimicrobial peptide with mucosal immune adjuvant activity, and Co1, an M-cell-targeting ligand. Intranasal administration of the recombinant PLpro antigen conjugate into C57BL/6 and hACE2 knock-in (KI) mice induced antigen-specific T-cell and antibody responses with complement-dependent cytotoxic activity. Viral challenge experiments using the Wuhan and Delta strains of SARS-CoV-2 provided further evidence that immunized hACE2 KI mice were protected against viral challenge infections. Our study shows that PLpro is a useful candidate vaccine antigen against SARS-CoV-2 infection and that the inclusion of human beta-defensin 2 and Co1 in the recombinant construct may enhance the efficacy of the vaccine.

TLR5 agonist in combination with anti-PD-1 treatment enhances anti-tumor effect through M1/M2 macrophage polarization shift and CD8+ T cell priming

Immune checkpoint inhibitors have revolutionized anti-tumor therapy, notably improving treatment responses in various tumors. However, many patients remain non-responsive and do not experience benefits. Given that Toll-like receptors (TLRs) can counteract tumor immune tolerance by stimulating both innate and adaptive immune responses, TLR agonists are being explored as potential immune adjuvants for cancer treatment. In this study, we assessed the potential of enhancing the efficacy of immune checkpoint inhibitors by activating innate immunity with a TLR5 agonist. In a mouse tumor model, combination therapy with TLR5 agonist and anti-PD-1 significantly inhibited tumor growth. The TLR5 agonist shifted the balance from M2-like to M1-like macrophages and upregulated the expression of co-stimulatory molecules in macrophages. Furthermore, TLR5 agonist promoted the activation and tumor infiltration of CD8+ T cells. As a result, the TLR5 agonist augmented the anti-tumor efficacy of anti-PD-1, suggesting its potential in modulating the tumor microenvironment to enhance the anti-tumor response. Our findings point toward the possibility of optimizing immune checkpoint inhibitor therapy using TLR5 agonists.

Increasing outer membrane complexity: the case of the lipopolysaccharide lipid A from marine Cellulophaga pacifica

Gram-negative bacteria living in marine waters have evolved peculiar adaptation strategies to deal with the numerous stress conditions that characterize aquatic environments. Among the multiple mechanisms for efficient adaptation, these bacteria typically exhibit chemical modifications in the structure of the lipopolysaccharide (LPS), which is a fundamental component of their outer membrane. In particular, the glycolipid anchor to the membrane of marine bacteria LPSs, i.e. the lipid A, frequently shows unusual chemical structures, which are reflected in equally singular immunological properties with potential applications as immune adjuvants or anti-sepsis drugs. In this work, we determined the chemical structure of the lipid A from Cellulophaga pacifica KMM 3664T isolated from the Sea of Japan. This bacterium showed to produce a heterogeneous mixture of lipid A molecules that mainly display five acyl chains and carry a single phosphate and a D-mannose disaccharide on the glucosamine backbone. Furthermore, we proved that C. pacifica KMM 3664T LPS acts as a weaker activator of Toll-like receptor 4 (TLR4) compared to the prototypical enterobacterial Salmonella typhimurium LPS. Our results are relevant to the future development of novel vaccine adjuvants and immunomodulators inspired by marine LPS chemistry.

A stress sensor, IRE1α, is required for bacterial-exotoxin-induced interleukin-1β production in tissue-resident macrophages

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1β (IL-1β), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1β production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1β production, indicating that IRE1α is required for CT- or CTB-induced IL-1β production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

Preparation and characterization of curdlan-chitosan conjugate nanoparticles as mucosal adjuvants for intranasal influenza H1N1 subunit vaccine

Intranasal vaccination offers crucial protection against influenza virus pandemics. However, antigens, especially subunit antigens, often fail to induce effective immune responses without the help of immune adjuvants. Our research has demonstrated that a polyelectrolyte complex, composed of curdlan sulfate/O-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (CS/O-HTCC), effectively triggers both mucosal and systemic immune responses when administrated intranasal. In this study, stable nanoparticles formed by curdlan-O-HTCC conjugate (CO NP) were prepared and characterized. Furthermore, the efficacy of CO NP was evaluated as a mucosal adjuvant in an intranasal influenza H1N1 subunit vaccine. The results revealed that CO NP exhibits uniform and spherical morphology, with a size of 190.53 ± 4.22 nm, and notably, it remains stable in PBS at 4 °C for up to 6 weeks. Biological evaluation demonstrated that CO NP stimulates the activation of antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), both in vitro and in vivo. Furthermore, intranasal administration of CO NP effectively elicits cellular and humoral immune responses, notably enhancing mucosal immunity. Thus, CO NP emerges as a promising mucosal adjuvant for influenza subunit vaccines.

Immunoantitumor Activity and Oxygenation Effect Based on Iron-Copper-Doped Folic Acid Carbon Dots.

Cancer metastasis and recurrence are closely associated with immunosuppression and a hypoxic tumor microenvironment. Chemodynamic therapy (CDT) and photothermodynamic therapy (PTT) have been shown to induce immunogenic cell death (ICD), effectively inhibiting cancer metastasis and recurrence when combined with immune adjuvants. However, the limited efficacy of Fenton's reaction and suboptimal photothermal effect present significant challenges for successfully inducing ICD through CDT and PTT. This paper described the synthesis and immunoantitumor activity of the novel iron-copper-doped folic acid carbon dots (CFCFB). Copper-doped folic acid carbon dots (Cu-FACDs) were initially synthesized via a hydrothermal method, using folic acid and copper gluconate as precursors. Subsequently, the nanoparticles CFCFB were obtained through cross-linking and self-assembly of Cu-FACDs with ferrocene dicarboxylic acid (FeDA) and 3-bromopyruvic acid (3BP). The catalytic effect of carbon dots in CFCFB enhanced the activity of the Fenton reaction, thereby promoting CDT-induced ICD and increasing the intracellular oxygen concentration. Additionally, 3BP inhibited cellular respiration, further amplifying the oxygen concentration. The photothermal conversion efficiency of CFCFB reached 55.8%, which significantly enhanced its antitumor efficacy through photothermal therapy. Immunofluorescence assay revealed that treatment with CFCFB led to an increased expression of ICD markers, including calreticulin (CRT) and ATP, as well as extracellular release of HMGB-1, indicating the induction of ICD by CFCFB. Moreover, the observed downregulation of ARG1 expression indicates a transition in the tumor microenvironment from an immunosuppressive state to an antitumor state following treatment with CFCFB. The upregulation of IL-2 and CD8 expression facilitated the differentiation of effector T cells, resulting in an augmented population of CD8+ T cells, thereby indicating the activation of systemic immune response.

Abstract 5761: A nanoplatform based on an in situ tumor vaccine and activation of STING and TLR7/8 pathways for enhanced NSCLC immunotherapy

Abstract Background: Immunotherapy has emerged as a promising treatment for tumors following surgical, radiotherapy, and chemotherapy interventions. Therapeutic tumor vaccines are considered highly effective in eliminating tumors and preventing their recurrence and metastasis due to their ability to induce or enhance T cell-mediated immune responses. However, the therapeutic efficacy of these vaccines is impeded by the heterogeneity present both between and within tumors. In contrast, in situ tumor vaccines, prepared from autologous antigens in tumor patients, present another promising strategy for generating comprehensive immune responses. Methods: In this study, we designed a multifunctional nanopolymer, the transformable nanoassembly PEG-Ce6@PAEMA/R848. This assembly includes the photosensitizer polyethylene glycol-chlorin-e6 (PEG-Ce6), the tumor pH-sensitive polymer poly(2-azepane ethyl methacrylate) (PAEMA), and the immune adjuvant Resiquimod (R-848). The objective is to confirm the activation of bone marrow-derived dendritic cells (DCs) at the cellular level. Subsequently, we investigated the maturation of DCs in lymph nodes and tumor sites in vivo. Results: In the particle characterization process, successful synthesis of nanoparticles was achieved, along with the verification of their acid-responsive function and toxic effects. Furthermore, in vitro experiments demonstrated the activation of bone marrow-derived dendritic cells (DCs) (Figure 1).To further investigate the in vivo vaccine effects, C57BL/6 mice were euthanized, and lymph nodes were extracted to examine the accumulation of FITC-PAEMA. It can be observed that, due to the reduction in size of the particles in the tumor acidic environment, the immune adjuvant can easily enter the lymph nodes. Activation of the STING pathway in dendritic cells within the lymph nodes was detected through qPCR. The maturation of dendritic cells (DCs) in the lymph nodes was determined using flow cytometry (Figure 2). Conclusions: In this study, we developed a straightforward yet potent strategy focused on in situ tumor targeting. This approach effectively inhibits tumor growth while inducing immunogenic cell death and eliciting a systemic anti-tumor immune response against cancer cells. Additionally, we intend to validate the therapeutic efficacy of this strategy on metastatic and distal tumors in subsequent investigations. Citation Format: Haiyu Zhou, Hengliang Hou, Bin Xu, Yucheng Huang, Hongrui Qiu. A nanoplatform based on an in situ tumor vaccine and activation of STING and TLR7/8 pathways for enhanced NSCLC 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 5761.

Abstract 4113: AST-301,a pDNA-based cancer vaccine encoding HER2-ICD, enhances anti-tumor effect of HER2-ADC in a HER2-expressed gastric cancer xenograft model

Abstract Background: In gastric cancer, the HER2 overexpression (6% to 29.5% of gastric cancers) is considered to increase proliferation activity and suppress apoptosis of the cancer cells. However, HER2-targeting therapy in gastric cancer has not been fully evaluated. Currently, new approaches with HER2-tageted antibody-drug conjugates (ADCs) have led to promising results in HER2-related solid tumors. ADCs may have immune-modulating properties and that the payloads delivered by ADCs may induce immunogenic cell death. Based on this rationale, ADC and cancer vaccine combination would be potentially synergistic to improve clinical outcomes. This study was conducted to evaluate an synergistic effect and immune response of the combination of AST-301 and HER2-ADC in HER2-expressed gastric cancer xenograft model. AST-301 phase 1 study (PN109) was completed and phase 2 randomized-control trials (CornerStone-001, CornerStone-003) have been conducted in breast cancer and gastric cancer. Methods: NCI-N87 cells were mixed with the same amount of matrigel at 1x107 cells to formation tumors in the flanks of athymic BALB/c nude mice. After the tumor size of mice reached 100-200 mm3, all of them were randomly classified into four groups (n=7/group) and drug administration was initiated. AST-301 (100 μg/animal, intradermal injection, mixed with rhuGM-CSF as an immune adjuvant) was administered once a week to a total of fourth. LCB01 (1 mg/kg, intravenous injection) was administered to once. Enhertu (1 or 3 mg/kg, intravenous injection) was administered to twice for four weeks. The relative tumor size and TGI rate at day 25 were key endpoints of efficacy. To evaluate the mechanism underlying the enhanced anti-tumor effect due to the combined administration of AST-301 and HER2-ADCs, immune-cell profiling was also conducted using FACS analysis. Results: Our previous study has shown that AST-301 has anti-tumor effects in human gastric cancer xenograft athymic BALB/c nude mouse model. It was showed that adding AST-301 to LCB01 or Enhertu inhibited tumor growth based on a TGI rate at days 25; AST-301 combining with LCB01 vs LCB01 mono (52 % vs 43 %), AST-301 combining with Enhertu vs Enhertu mono (42 % vs 24 %).As an explorative endpoint, it was observed that myeloid-derived suppressor cells (MDSCs) elevation was modestly inhibited in tumor site in AST-301 mono or certain combination regimen adding AST-301. It was remarkable (or observed or captured) that MDSCs elevation was inhibited in tumor site in the combination groups of all ADCs with AST-301 compared to normal saline groups. Conclusion: With combining AST-301 and HER2 ADCs, it was demonstrated that anti-tumor effect was likely synergistic in the athymic mouse model. Phase 2 study of AST-301 in HER2-expressed gastric cancer has been on going (CornerStone-003, NCT 05771584). Citation Format: Jinho Kang, Hyo-Hyun Park, Jee Hyun Choi, Jinback Lim, Seong-Yong Jang, Min-Ah Kim, Myeong-Kyu Park, Yun-Hee Park, Soyeon Lim, Chul-Woong Chung, Jin Kyeong Choi, Eunkyo Joung, Ashley Yongmin Kim, Hun Jung. AST-301,a pDNA-based cancer vaccine encoding HER2-ICD, enhances anti-tumor effect of HER2-ADC in a HER2-expressed gastric cancer xenograft 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 4113.

Abstract 4114: AST-201 (pUMVC3-hIGFBP2 N-terminus) demonstrates anti-tumor effect in an ovarian cancer mouse model

Abstract Background: IGFBP2, known to enhance the invasion capacity of ovarian cancer cells, has been suggested that its inhibition could be potentially a treatment strategy of ovarian cancer. AST-201 (pUMVC3-hIGFBP2) is a therapeutic cancer vaccine using a plasmid DNA encoding IGFBP2 N-terminus. In a phase 1 study (NCT01322802) completed, 100 μg AST-201 (intradermal immunization, id) showed not only a significant efficacy by inducing the Th1-cell immunity against IGFBP2 but also safety and tolerability profiles in ovarian cancer patients. The primary objective of this study is to evaluate whether the administration of AST-201 alone and the combination with pembrolizumab could show anti-tumor efficacy and/or synergic effect in the ID8-Luc2 ovarian cancer mouse model. Also, immunological responses were observed as explorative endpoint. Methods: AST-201 (100 μg/animal, id, mixed with mGM-CSF as an immune adjuvant) was immunized to mice (C57BL/6) once a week for a total of 4 times on different days, either alongside pembrolizumab (10 mg/kg, intraperitoneal injection) twice a week for a total of 3 times on different days. Also, AST-201 was immunized was a mono treatment. The efficacy was evaluated by a tumor growth inhibition (TGI) rate at the last day, and immune cell profiling via FACS analysis was conducted with splenocytes and tumor tissues collected at 8 weeks after the first injection. Results: As a primary endpoint, a TGI rate at Day 55 of AST-201 mono treatment was 67%, comparing to a control group (p<0.05). The anti-tumor effect of AST-201 combining with pembrolizumab was better than standard dose pembrolizumab, based on a TGI rate at Day 55 (78% vs 66%, not significant). Immune profiling showed that AST-201 and pembrolizumab combination regimen could inhibit a tumor growth by transforming TME into inflamed-type (‘hot’) form the low immunoreactivity, which was supported by increased CD4+TEM, and CD8+TEM and T helper cells in splenocyte and TIL analysis. Conclusion: A study demonstrated that AST-201 (IGFBP2 cancer vaccine) showed an anti-tumor effect as mono treatment and would be potentially leading to a synergistic effect with a combination regimen of pembrolizumab. Phase 2 randomized-controlled study of AST-201 in ovarian cancer will be initiated under the MRCT strategy (CornerStone-004 study, NCT05794659). Citation Format: Jinback Lim, Jinho Kang, Hyo-Hyun Park, Jin Kyeong Choi, Jee Hyun Choi, Seong-Yong Jang, Min-Ah Kim, Myeong-Kyu Park, Mary L Disis, Eunkyo Joung, Ashley Yongmin Kim, Hun Jung. AST-201 (pUMVC3-hIGFBP2 N-terminus) demonstrates anti-tumor effect in an ovarian cancer mouse 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 4114.

Abstract 4107: Evaluating the efficacy of personalized neoantigen cancer vaccine in preclinical model: A promising step towards precision immunotherapy

Abstract Background: In the past decade, immunotherapy has transformed cancer treatment, with the development of immune-checkpoint inhibitors and adoptive cell therapies. A novel frontier is personalized vaccines targeting neoantigens unique to tumor cells. Vaccines based on neoantigens have several advantages. First, neoantigens are exclusively expressed by tumor cells and can, therefore, elicit truly tumor-specific T cell responses, thereby preventing ‘off-target’ damage to nonmalignant tissues. Second, neoantigens are de novo epitopes derived from somatic mutations, which presents the possibility to circumvent T cell central tolerance of self-epitopes and thus induce immune responses to tumors. One of the major challenges is the optimal neoantigen discovery/selection. Our study combines in silico HLA binding models with the 'MHC-PepSeq' platform to enhance neoantigen prediction, creating a personalized neoantigen peptide vaccine. We aim to demonstrate its efficacy in a mouse CT26 colon cancer model. Methods: Our method involved whole exome sequencing of tumor and normal DNA to identify neoantigens expressed in CT26 tumors, generating a peptide library. The peptides were further filtered using an in-silico prediction algorithm and our proprietary peptide-MHC binding assay (PepSeq assay), to determine those exhibiting the highest binding affinity with mice HLAs. Vaccines were prepared by dissolving these peptides in sterile PBS. Mice (BALB/C) were subcutaneously implanted with CT26 tumors, then immunized with the vaccine via IV injection in combination with a TLR7/8 agonist. Mice received a second vaccine dose on Day 14, and some groups were treated with an anti-PD-1 agent. Tumors and spleens from the mice were collected, processed into single-cell suspensions, and analyzed by single-cell sequencing, TCR analysis, IHC, and flow cytometry assays. Peripheral blood mononuclear cells (PBMCs) were collected and utilized in MHC tetramer assays. Results: Preliminary in vivo studies demonstrated significant tumor growth restrictions (TGI around 70%) and prolonged tumor-free survival in some mice. Our data showed the generation of neoantigen-specific T cells in vaccinated mice. Remarkably, vaccinated, tumor-free mice, when re-challenged with the same tumor cells, maintained their tumor-free status, suggesting the vaccine induced post-treatment immunological memory. Combining the vaccine with checkpoint inhibition resulted in better tumor growth restriction in mice (TGI around 93%). Conclusion: Overall, our data suggests that our personalized neoantigen vaccine platform has the potential to evoke robust and durable immune responses and, when paired with suitable complementary therapies and immune adjuvants, may serve as an effective cancer treatment. Citation Format: Tithi Ghosh Halder, Kate Gutowsky, Serina Ng, Trason Thode, Alexis Weston, Erin Kelley, Jorge Soria-Bustos, Jorge A. Giron, Taylor Bargenquast, Mohan Kaadige, Sanjana Tripuraneni, Brian Durbin, Shelby Rheinschmidt, Sydney Adamson, Michael Gordon, Justin Moser, John Altin, Raffaella Soldi, Sunil Sharma. Evaluating the efficacy of personalized neoantigen cancer vaccine in preclinical model: A promising step towards precision 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 4107.

Abstract 5262: MBO enhances anti-tumor immunity in pancreatic ductal adenocarcinoma by inducing immunogenic apoptosis

Abstract Pancreatic ductal adenocarcinoma (PDAC) poses significant therapeutic challenges due to its aggressive nature and limited treatment options. In this study, we explored the anti-cancer potential of MBO, a compound selected through drug repurposing strategy based on its cytotoxic ability and modulation of key survival pathways in PDAC. Our preliminary in vitro experiments demonstrated MBO's potent cytotoxicity against various PDAC cell lines, inhibiting cellular growth and clonogenicity. MBO induced G1 phase cell cycle arrest in a concentration-dependent manner. In the next step, in vivo, studies using xenograft and orthotopic mouse models confirmed MBO's ability to suppress tumor growth and increase CD8+ T cell infiltration. MBO demonstrated a favorable tumor suppressive activity wherein it suppressed tumor growth by approximately 49% and 56% in xenograft and orthotopic models, respectively. A CD8+ T cell depletion experiment revealed partial dependence on CD8+ T cells for MBO's overall anti-tumor efficacy. Further, combining MBO with anti-PD1 therapy exhibited a synergistic effect, enhancing overall T cell infiltration, particularly CD8+ T cells. Our mechanistic studies revealed that MBO induced immunogenic apoptosis, as evidenced by increased expression of immunogenic cell death markers (HSP90 and calreticulin) and apoptotic markers. To further delineate the mechanism, we performed RPPA profiling. RPPA analysis indicated MBO's impact on mitochondrial gene expression and stress response pathways, suggesting its involvement in disrupting cellular survival mechanisms along with stress-induced immunogenic marker expression. Overall, this study demonstrates MBO as a promising anti-cancer agent with the potential to serve as an immune adjuvant. Particularly, in combination with immunotherapies, MBO can serve as a potential option to improve immunotherapeutic response in PDAC tumors providing valuable insights for developing novel therapeutic strategies in PDAC. Citation Format: Shreyas Ramchandra Gaikwad, Sanjay K. Srivastava. MBO enhances anti-tumor immunity in pancreatic ductal adenocarcinoma by inducing immunogenic apoptosis [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 5262.

Abstract 5461: Antecedent STING agonist therapy improves tumor response to chemotherapy in murine models of osteosarcoma

Abstract Little improvement has been made in outcomes for pediatric patients with osteosarcoma in decades. Cytotoxic chemotherapy alone is insufficient for cure and upfront complete surgical resection is usually required to achieve long-term survival. Better systemic therapies are needed. Osteosarcoma is characterized by a high degree of genomic instability that would suggest these tumors have sufficient neoantigen burden to be immunogenic and responsive to immunotherapy, but trials of immune checkpoint inhibitors in osteosarcoma have shown poor effectiveness. Although immunotherapy drugs targeting the adaptive immune system have worked poorly, we hypothesize that an innate immune adjuvant - stimulator of interferon response cGAMP interactor 1 (STING) agonist - in combination with cytotoxic chemotherapy would improve chemotherapy effectiveness and immune-mediated tumor clearance, particularly given the abundance of innate immune cells in the microenvironment of these tumors. Using two different immune-competent in vivo murine models of osteosarcoma (K7M2 in BALB/c mice and F420 in C57BL/6 mice), we challenged mice with tumor cells via subcutaneous or intratibial injection. In all experimental replicates, we observed improved tumor regression in mice treated with intratumoral STING agonist followed by intraperitoneal treatment with carboplatin. Responses included complete tumor response in up to 40% of treated mice. Treatment with STING agonist alone or carboplatin alone only delayed tumor growth compared to untreated controls. Additionally, mice treated first with carboplatin and then with STING agonist demonstrated an initial modest response to treatment, but ultimately exhibited tumor progression, suggesting an importance to the sequence of STING agonist administration. In vitro treatment of K7M2 and F420 cells with STING agonist showed a tumor-intrinsic transient increase in expression of pro-inflammatory mediators Cxcl10, Ccl5, and Ifnb 4 hours after STING exposure. Flow cytometry analysis of K7M2 tumors demonstrated increased infiltration of STING/carboplatin-treated tumors with CD8+/IFNγ+ lymphocytes and CD11b+/MHCII+/F480+ tumor-associated macrophages. Interestingly, these tumors also demonstrated an increased quantity of CD11b+/MHCII-/F480-/Ly6Glo/Ly6C+ myeloid-derived suppressor cells despite their favorable clinical response. Our findings suggest that intratumoral STING agonist therapy stimulates tumor-intrinsic and tumor-extrinsic pro-inflammatory mediators that improve the tumor response to treatment with carboplatin. Ongoing studies are further investigating the tumor-intrinsic and extrinsic mediators that are driving this improved response to therapy. Citation Format: Erin E. Resch, Emily Kulp, Michele Doucet, Alice Recho, Brian H. Ladle. Antecedent STING agonist therapy improves tumor response to chemotherapy in murine models of osteosarcoma [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 5461.

Abstract 1422: A novel gut microbiota therapeutic approach for enhancing immune checkpoint inhibitor therapy

Abstract Immune checkpoint inhibitor therapy (ICT) has revolutionized cancer care, but up to 50% of patients do not respond to ICT. A host factor implicated in variable ICT response is the gut microbiome: a) germfree/antibiotic-treated mice do not respond to ICT; b) human ICT responders have distinct gut microbiome signatures compared to non-responders; and c) fecal microbiota transplants are safe and potentially effective in cancer patients previously unresponsive to ICT. We have previously established that ICT induces translocation of gut microbiota into secondary lymphoid tissues (particularly the mesenteric lymph nodes) and tumor which is required to enhance extraintestinal anti-tumor immunity (PMID: 36867675). Here, we have developed a novel gut microbiota therapy (GMT) which circumvents the challenges and limitations of conventional microbiome therapies that use live microbes. GMT utilizes specific components derived from commensal gut microbiota identified in our published clinical study (PMID:28923537) that can be administered parenterally (subcutaneous/intravenous) to safely and reproducibly enhance ICT efficacy in multiple preclinical cancer models (B16-F10 with anti-PD-1 and/or CTLA-4, MC38 with or without anti-PD-1, humanized mouse with human melanoma and human anti-PD-1). GMT acts by agonizing the pattern recognition receptors hTLR1/2/6, hNOD2 and hSTING. GMT induces anti-tumor cytokines (IL-12) but does not significantly increase sepsis associated cytokines (IL-6, TNFa) in both human immune cells and in non-human primates. Mechanistically, GMT acts via dendritic cells, and potentially through increased expression of delta-like ligand protein 4 (DLL4), a Notch signaling ligand that activates Notch 1/4 in T cells and promotes Th1 and Th17 differentiation. Current efforts to identify the active components of GMT are underway in which we have used serial liquid-liquid extractions (ethyl acetate followed by water:methanol:chloroform) and subsequent reverse phase chromatography to identify the most active fractions and identification of the bacterial components/constituents within these fractions. Together, these results demonstrate that GMT has the potential to serve as an innate immune adjuvant (akin to vaccine adjuvants) that enhances the effect of immunotherapy for possibly many cancer types. Citation Format: Priscilla Del Valle, Gabriella Nguyen, Yongbin Choi, Parastoo Sabaeifard, Wenng li, Jake Lichterman, Laura Coughlin, Nicole Poulides, Angelica Sanjuan, Andrew Y. Koh. A novel gut microbiota therapeutic approach for enhancing immune checkpoint inhibitor therapy [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 1422.

Exploring Immunological Effects and Novel Immune Adjuvants in Immunotherapy for Salivary Gland Cancers.

Salivary gland cancer (SGC) is rare and comprises over 20 histological subtypes. Recently, clinical experience regarding immunotherapies for SGCs has been accumulating, yet their efficacy remains controversial. Understanding the tumor microenvironment (TME), including the expression of immune checkpoint molecules in SGC, is crucial to optimizing immunotherapy. In this review, we demonstrate that high-grade mucoepidermoid carcinoma and salivary duct carcinoma generally exhibit immune-hot TME with high immune cell infiltration, frequent genetic mutations, and robust immune checkpoint molecule expression. In contrast, adenoid cystic carcinomas exhibit an immune-cold TME. While the reported efficacy of immune checkpoint inhibitors (ICIs) for SGCs is generally poor, several studies showed promising clinical efficacy of ICIs, with an objective response rate ranging from 20.0-33.3%, indicating that ICIs might be beneficial for a specific population of SGC. Molecule-targeted therapies including anti-human epidermal growth factor receptor 2 and anti-androgen receptor therapies have shown promising clinical efficacy against SGC. Recent evidence indicates that these molecules could be targets for antigen-specific immunotherapies including chimeric antigen receptor-T therapy and cancer vaccines. This review discusses the current understanding and future directions of immunotherapies for SGCs, including ongoing clinical trials.

Differential Immune Response Patterns Induced by Anionic and Cationic Lipid Adjuvants in Intranasal Anti-Influenza Immunization.

Currently, vaccine development against different respiratory diseases is at its peak. It is of utmost importance to find suitajble adjuvants that can increase the potency of the vaccine candidates. This study aimed to determine the systemic and splenic immune mechanisms in mice models induced by anionic and cationic lipid adjuvants in the presence of the vaccine-candidate influenza antigen hemagglutinin (HA). In the presence of the HA antigen, the cationic adjuvant (N3) increased conventional dendritic cell 1 (cDC1) abundance with enhanced MHCI and CD80-CD86 costimulatory marker expression, and significantly higher CD8T and Th17 populations with enhanced interferon-gamma (IFNγ) expression in CD8T and CD4T populations. Conversely, the anionic adjuvant (L3) increased the cDC2 population percentage with significantly higher MHCII and DEC205 expression, along with an increase in the CD4T and regulatory T cell populations. The L3-treated group also exhibited higher percentages of activated B and plasma cell populations with significantly higher antigen-specific IgG and IgA titer and virus neutralization potential. While the anionic adjuvant induced significantly higher humoral responses than the cationic adjuvant, the latter influenced a significantly higher Th1/Th17 response. For customized vaccine development, it is beneficial to have alternative adjuvants that can generate differential immune responses with the same vaccine candidate antigen. This study will aid the selection of adjuvants based on their charges to improve specific immune response arms in the future development of vaccine formulation.

Overcoming neutrophil-induced immunosuppression in postoperative cancer therapy: Combined sialic acid-modified liposomes with scaffold-based vaccines

Immunotherapy is a promising approach for preventing postoperative tumor recurrence and metastasis. However, inflammatory neutrophils, recruited to the postoperative tumor site, have been shown to exacerbate tumor regeneration and limit the efficacy of cancer vaccines. Consequently, addressing postoperative immunosuppression caused by neutrophils is crucial for improving treatment outcomes. This study presents a combined chemoimmunotherapeutic strategy that employs a biocompatible macroporous scaffold-based cancer vaccine (S-CV) and a sialic acid (SA)-modified, doxorubicin (DOX)-loaded liposomal platform (DOX@SAL). The S-CV contains whole tumor lysates as antigens and imiquimod (R837, Toll-like receptor 7 activator)-loaded PLGA nanoparticles as immune adjuvants for cancer, which enhance dendritic cell activation and cytotoxic T cell proliferation upon localized implantation. When administered intravenously, DOX@SAL specifically targets and delivers drugs to activated neutrophils in vivo, mitigating neutrophil infiltration and suppressing postoperative inflammatory responses. In vivo and vitro experiments have demonstrated that S-CV plus DOX@SAL, a combined chemo-immunotherapeutic strategy, has a remarkable potential to inhibit postoperative local tumor recurrence and distant tumor progression, with minimal systemic toxicity, providing a new concept for postoperative treatment of tumors.

Lymph-targeted high-density lipoprotein-mimetic nanovaccine for multi-antigenic personalized cancer immunotherapy.

Cancer vaccines show huge potential for cancer prevention and treatment. However, their efficacy remains limited due to weak immunogenicity regarding inefficient stimulation of cytotoxic T lymphocyte (CTL) responses. Inspired by the unique characteristic and biological function of high-density lipoprotein (HDL), we here develop an HDL-mimicking nanovaccine with the commendable lymph-targeted capacity to potently elicit antitumor immunity using lipid nanoparticle that is co-loaded with specific cancer cytomembrane harboring a collection of tumor-associated antigens and an immune adjuvant. The nanoparticulate impact is explored on the efficiency of lymphatic targeting and dendritic cell uptake. The optimized nanovaccine promotes the co-delivery of antigens and adjuvants to lymph nodes and maintains antigen presentation of dendritic cells, resulting in long-term immune surveillance as the elevated frequency of CTLs within lymphoid organs and tumor tissue. Immunization of nanovaccine suppresses tumor formation and growth and augments the therapeutic efficacy of checkpoint inhibitors notably on the high-stemness melanoma in the mouse models.

Exploration of the Binding Mechanism of Cyclic Dinucleotide Analogs to Stimulating Factor Proteins and the Implications for Subsequent Analog Drug Design.

Cyclic dinucleotides (CDNs) are cyclic molecules consisting of two nucleoside monophosphates linked by two phosphodiester bonds, which act as a second messenger and bind to the interferon gene stimulating factor (STING) to activate the downstream signaling pathway and ultimately induce interferon secretion, initiating an anti-infective immune response. Cyclic dinucleotides and their analogs are lead compounds in the immunotherapy of infectious diseases and tumors, as well as immune adjuvants with promising applications. Many agonists of pathogen recognition receptors have been developed as effective adjuvants to optimize vaccine immunogenicity and efficacy. In this work, the binding mechanism of human-derived interferon gene-stimulating protein and its isoforms with cyclic dinucleotides and their analogs was theoretically investigated using computer simulations and combined with experimental results in the hope of providing guidance for the subsequent synthesis of cyclic dinucleotide analogs.

The effect of lentinan on dexamethasone-induced immunosuppression in mice

The immune system acts as a vital defense barrier against pathogenic invasions, and its stable operation is crucial for maintaining body health. Nevertheless, various natural or artificial factors can compromise the body's immune function, leading to immunosuppression, which may interfere with the efficacy of vaccination and increase the susceptibility of the body to disease-causing pathogens. In an effort to ensure successful vaccinations and improve overall physical well-being, the search for appropriate immune regulators to enhance immunity is of paramount importance. Lentinan (LNT) has a significant role in immune regulation and vaccine adjuvants. In the present study, we constructed an immunosuppressive model using dexamethasone (DEX) and demonstrated that LNT could significantly improved antibody levels in immunosuppressive mice and stimulated T-lymphocyte proliferation and differentiation in intestinal Peyer's patches. LNT also increased the production of secretory immunoglobulin A (sIgA) in the duodenal fluid, the number of goblet cells, and the proportion of mucin area. Moreover, LNT modulated the intestinal microbiota and increased the production of short-chain fatty acids. Additionally, LNT promoted the proliferation, differentiation, and pro-inflammatory cytokines production of DEX-treated splenic T lymphocytes in vitro. Thus, the present study highlights the potential of LNT in reversing immunosuppression and avoiding the failure of vaccination.

Intranasal Drug Delivery of Antiviral Agents - A Revisit and Way Forward

Abstract: Even to this date, oral drug delivery in the form of tablets, capsules, and syrups is considered as the most accepted one. However, oral delivery as a methodology requires that the active molecules and their formulations are water-soluble. Nasal drug delivery is characterized by ease of permeability through the epithelial mucosa, low enzyme activity, and a wide range of immunocompetent cells. For the transfer of drugs and active molecules through the nasal route, it is often essential to resort to nanodelivery methods, such as liposomes, microspheres, nanoemulsions, and so on. The use of nanodelivery vehicles has become more important in the modern context of viral infections, including those of the respiratory tract. Nanoformulations are developed in the form of nasal gels, sprays, drops, rinses, etc. Nanoformulations of antigens, vaccine and immune adjuvants, and antivirals are now gaining importance. There are promising reports on nanoparticles of metals, metal oxides, polymers, and so on that have the potential to detect and inhibit viruses by themselves. This review looks into the nasal nanoformulations in detail and provides an insight into how their efficacy can be improved. To overcome known drawbacks, such as degradation and active mucociliary clearance by antigenpresenting cells at the site of administration, polymers, such as PEG, are incorporated in the nanoformulation. Polymeric systems also provide better tunability of physicochemical properties. The mechanism of nasal spray-based drug delivery systems is also discussed in this paper. The review, thus, provides a detailed insight into the way forward for the development of nasal formulations.