Enhanced Antigen Presentation Research Articles

Myeloid-specific KDM6B inhibition sensitizes glioblastoma to PD1 blockade.

Glioblastoma (GBM) tumors are enriched in immune-suppressive myeloid cells and are refractory to immune checkpoint therapy (ICT). Targeting epigenetic pathways to reprogram the functional phenotype of immune-suppressive myeloid cells to overcome resistance to ICT remains unexplored. Single-cell and spatial transcriptomic analyses of human GBM tumors demonstrated high expression of an epigenetic enzyme-histone 3 lysine 27 demethylase (KDM6B)-in intratumoral immune-suppressive myeloid cell subsets. Importantly, myeloid cell-specific Kdm6b deletion enhanced proinflammatory pathways and improved survival in GBM tumor-bearing mice. Mechanistic studies showed that the absence of Kdm6b enhances antigen presentation, interferon response and phagocytosis in myeloid cells by inhibition of mediators of immune suppression including Mafb, Socs3 and Sirpa. Further, pharmacological inhibition of KDM6B mirrored the functional phenotype of Kdm6b-deleted myeloid cells and enhanced anti-PD1 efficacy. This study thus identified KDM6B as an epigenetic regulator of the functional phenotype of myeloid cell subsets and a potential therapeutic target for enhanced response to ICT.

LAMP1 targeting of the large T antigen of Merkel cell polyomavirus results in potent CD4 T cell responses and tumor inhibition.

Most cases of Merkel cell carcinoma (MCC), a rare and highly aggressive type of neuroendocrine skin cancer, are associated with Merkel cell polyomavirus (MCPyV) infection. MCPyV integrates into the host genome, resulting in expression of oncoproteins including a truncated form of the viral large T antigen (LT) in infected cells. These oncoproteins are an attractive target for a therapeutic cancer vaccine. We designed a cancer vaccine that promotes potent, antigen-specific CD4 T cell responses to MCPyV-LT. To activate antigen-specific CD4 T cells in vivo, we utilized our nucleic acid platform, UNITE™ (UNiversal Intracellular Targeted Expression), which fuses a tumor-associated antigen with lysosomal-associated membrane protein 1 (LAMP1). This lysosomal targeting technology results in enhanced antigen presentation and potent antigen-specific T cell responses. LTS220A, encoding a mutated form of MCPyV-LT that diminishes its pro-oncogenic properties, was introduced into the UNITE™ platform. Vaccination with LTS220A-UNITE™ DNA vaccine (ITI-3000) induced antigen-specific CD4 T cell responses and a strong humoral response that were sufficient to delay tumor growth of a B16F10 melanoma line expressing LTS220A. This effect was dependent on the CD4 T cells' ability to produce IFNγ. Moreover, ITI-3000 induced a favorable tumor microenvironment (TME), including Th1-type cytokines and significantly enhanced numbers of CD4 and CD8 T cells as well as NK and NKT cells. Additionally, ITI-3000 synergized with an α-PD-1 immune checkpoint inhibitor to further slow tumor growth and enhance survival. These findings strongly suggest that in pre-clinical studies, DNA vaccination with ITI-3000, using the UNITE™ platform, enhances CD4 T cell responses to MCPyV-LT that result in significant anti-tumor immune responses. These data support the initiation of a first-in-human (FIH) Phase 1 open-label study to evaluate the safety, tolerability, and immunogenicity of ITI-3000 in patients with polyomavirus-positive MCC (NCT05422781).

Tumor immunogenicity dictates reliance on TCF1 in CD8+ T cells for response to immunotherapy

Stem-like CD8+ Tcells are regulated by Tcell factor 1 (TCF1) and are considered requisite for immune checkpoint blockade (ICB) response. However, recent findings indicate that reliance on TCF1+CD8+ Tcells for ICB efficacy may differ across tumor contexts. We find that TCF1 is essential for optimal priming of tumor antigen-specific CD8+ Tcells and ICB response in poorly immunogenic tumors that accumulate TOX+ dysfunctional Tcells, but is dispensable for Tcell priming and therapy response in highly immunogenic tumors that efficiently expand transitory effectors. Importantly, improving Tcell priming by vaccination or by enhancing antigen presentation on tumors rescues the defective responses of TCF1-deficient CD8+ Tcells upon ICB in poorly immunogenic tumors. Our study highlights TCF1's role during the early stages of anti-tumor CD8+ Tcell responses with important implications for guiding optimal therapeutic interventions in cancers with low TCF1+CD8+ Tcells and low-neo-antigen expression.

Tong-Xie-Yao-Fang promotes dendritic cells maturation and retards tumor growth in colorectal cancer mice with chronic restraint stress

Ethnopharmacological relevanceDepression is one of the important risk factors that accelerate the progression of colorectal cancer (CRC). Tong-Xie-Yao-Fang (TXYF) is a widely used classical formula for treating psychiatric-related intestinal diseases in traditional Chinese medicine, that is composed of four different herbs: Atractylodes macrocephala Koidz. (Baizhu), Paeonia lactiflora Pall. (Baishaoyao), Citrus reticulata Blanco (Chenpi), Saposhnikovia divaricata (Turcz.) Schischk (Fangfeng). TXYF has over a hundred years of history and can significantly improve depression and reduce intestinal symptoms. However, the intervention effect and mechanism of TXYF on colorectal cancer accompanied by psychological stress are not still clear. Aim of studyThis study investigated the therapeutic effect of TXYF on CRC mice with chronic restraint stress (CRS) and to explore its mechanism. Materials and methodsWe constructed a mouse model of chronic stress by CRS and subcutaneous injection of CT26-Luc cells, and administered TXYF by gavage. We measured the body weight, tumor size, and tumor weight of each group of mice. The tumor growth was monitored dynamically of by vivo bioluminescence analysis. The depressive state of each group of mice were evaluated by tail suspension test, forced swimming test, and hormone level changes. We used flow cytometry to detect the ratio of CD4+ T cells, CD8+ T cells, Th1 cells, Th2 cells, and dendritic cells (DCs) phenotype (MHC II, CD80, and CD86) and chemotaxis ability (CXCR4 and CCR7) of in peripheral blood and tumor tissue. the levels of IL-12, IL-18, Th1 cytokines, and Th2 cytokines in the serum of each group of mice were determined by ELISA. ResultsTXYF can improve the body weight of CRC mice with CRS, inhibit tumor volume and weight, alleviate depressive state, upregulate 5-HT levels, and inhibit HPA axis hormone secretion. The results of flow cytometry showed that TXYF can promote the maturation of DCs phenotype and function, enhance antigen presentation ability, increase the ratio of CD4+ T cells and CD4+/CD8+ T cells, and shift Th1/Th2 balance towards Th1 cells, thus increasing serum levels of IFN-γ, IL-18, IL-2, and IL-12, while decreasing serum levels of IL-4 and IL-10, and effectively triggering T cell-mediated immune response. ConclusionsThis study shows that TXYF inhibits the growth of tumors in CRC mice with CRS by stimulating immune response. The mechanism may be inhibiting the HPA axis and promoting DCs maturation, thus activating T cells and enhancing anti-tumor immune response, ultimately preventing the progression of CRC.

Engineered Extracellular Vesicles to Enhance Antigen Presentation for Boosting Light-Driven Tumor Immunotherapy.

Extracellular vesicles (EVs) have emerged as potential tools for tumor-target therapy accompanied with activating anticancer immune responses by serving as an integrated platform, but usually suffered from the limited cross presentation of tumor-associated antigen by dendritic cells (DCs). Here, a straightforward engineering strategy to construct heat shock proteins 70 (HSP70) highly expressed EVs incapsulated with Te nanoparticles (Te@EVsHSP70 ) for tumor photothermal therapy triggering improved immunotherapy is proposed. Tumor cells are firstly used as bioreactors for intracellular synthesis of Te nanoparticles, and NIR irradiation is subsequently introduced to upregulate the expression of HSP70 to give engineered Te@EVsHSP70 through exocytosis. Te@EVsHSP70 exhibits excellent photothermal performance and enhanced tumor antigen capture capability, which induces significant immunogenic death of tumor cells and improves DCs maturation both in vitro and in vivo. Thus, the engineered EVs demonstrate superior antitumor efficacy through photothermal effect and following provoked antitumor immune responses. This work provides a facile method to fabricate multifunctional EVs-based drug delivery system for improving photothermal-triggered tumor immunotherapy.

Radiation dose, schedule, and novel systemic targets for radio-immunotherapy combinations.

Immunotherapy combinations are being investigated to expand the benefit of immune checkpoint blockade across many cancer types. Radiation combinations, in particular using stereotactic body radiotherapy, are of keen interest because of underlying mechanistic rationale, safety, and availability as a standard of care in certain cancers. In addition to direct tumor cytotoxicity, radiation therapy has immunomodulatory effects such as induction of immunogenic cell death, enhancement of antigen presentation, and expansion of the T-cell receptor repertoire as well as recruitment and increased activity of tumor-specific effector CD8+ cells. Combinations of radiation with cytokines and/or chemokines and anti-programmed death 1 and anticytotoxic T-lymphocyte antigen 4 therapies have demonstrated safety and feasibility, as well as the potential to improve long-term outcomes and possibly induce out of irradiated field or abscopal responses. Novel immunoradiotherapy combinations represent a promising therapeutic approach to overcome radioresistance and further enhance systemic immunotherapy. Potential benefits include reversing CD8+ T-cell exhaustion, inhibiting myeloid-derived suppressor cells, and reversing M2 macrophage polarization as well as decreasing levels of colony-stimulating factor-1 and transforming growth factor-β. Here, we discuss current data and mechanistic rationale for combining novel immunotherapy agents with radiation therapy.

Polymeric nanoparticles for DNA vaccine-based cancer immunotherapy: a review.

Cancer is one of the leading causes of death and mortality in the world. There is an essential need to develop new drugs or therapeutic approaches to manage treatment-resistant cancers. Cancer immunotherapy is a type of cancer treatment that uses the power of the body's immune system to prevent, control, and eliminate cancer. One of the materials used as a vaccine in immunotherapy is DNA. The application of polymeric nanoparticles as carriers for DNA vaccines could be an effective therapeutic approach to activate immune responses and increase antigen presentation efficiency. Various materials have been used as polymeric nanoparticles, including: chitosan, poly (lactic-co-glycolic acid), Polyethylenimine, dendrimers, polypeptides, and polyesters. Application of these polymer nanoparticles has several advantages, including increased vaccine delivery, enhanced antigen presentation, adjuvant effects, and more sustainable induction of the immune system. Besides many clinical trials and commercial products that were developed based on polymer nanoparticles, there is still a need for more comprehensive studies to increase the DNA vaccine efficiency in cancer immunotherapy using this type of carrier.

Development and anticancer properties of Up284, a spirocyclic candidate ADRM1/RPN13 inhibitor.

Bortezomib has been successful for treatment of multiple myeloma, but not against solid tumors, and toxicities of neuropathy, thrombocytopenia and the emergence of resistance have triggered efforts to find alternative proteasome inhibitors. Bis-benzylidine piperidones such as RA190 covalently bind ADRM1/RPN13, a ubiquitin receptor that supports recognition of polyubiquitinated substrates of the proteasome and their subsequent deububiqutination and degradation. While these candidate RPN13 inhibitors (iRPN13) show promising anticancer activity in mouse models of cancer, they have suboptimal drug-like properties. Here we describe Up284, a novel candidate iRPN13 possessing a central spiro-carbon ring in place of RA190's problematic piperidone core. Cell lines derived from diverse cancer types (ovarian, triple negative breast, colon, cervical and prostate cancers, multiple myeloma and glioblastoma) were sensitive to Up284, including several lines resistant to bortezomib or cisplatin. Up284 and cisplatin showed synergistic cytotoxicity in vitro. Up284-induced cytotoxicity was associated with mitochondrial dysfunction, elevated levels of reactive oxygen species, accumulation of very high molecular weight polyubiquitinated protein aggregates, an unfolded protein response and the early onset of apoptosis. Up284 and RA190, but not bortezomib, enhanced antigen presentation in vitro. Up284 cleared from plasma in a few hours and accumulated in major organs by 24 h. A single dose of Up284, when administered to mice intra peritoneally or orally, inhibited proteasome function in both muscle and tumor for >48 h. Up284 was well tolerated by mice in repeat dose studies. Up284 demonstrated therapeutic activity in xenograft, syngeneic and genetically-engineered murine models of ovarian cancer.

Phase I study of the bifunctional anti-PD-L1/TGF-βRII agent SHR-1701 combined with SHR2554, an EZH2 inhibitor, in patients with previously treated advanced lymphoma and solid tumors.

2507 Background: Despite the remarkable successes of cancer immunotherapies, the majority of patients (pts) experience only partial response and most pts will progress. Enhancer of zeste homolog 2 (EZH2) as an epigenetic target for cancer therapy has been shown to enhanced antigen presentation and synergize with immunotherapy in preclinical studies. This study investigates the safety and efficacy of SHR-1701 (anti-PD-L1/TGF-βRII) plus SHR2554 (EZH2i) in pts with pretreated advanced lymphoma and solid tumors. Methods: This open-label, single-arm, phase 1 study enrolled pts with pretreated advanced lymphoma and solid tumors. Escalating doses of SHR2554 (300–350mg bid) plus fixed-dose SHR-1701 (30mg/kg q3w) were evaluated in the dose-escalation phase. SHR2554 at the recommended phase 2 dose (RP2D) plus SHR-1701 were administered in the dose-expansion phase. The primary end point was determination of the RP2D of SHR2554 plus SHR-1701. Secondary end points included safety and preliminary activity. Results: By January 6, 2023, 33 patients were enrolled (dose-escalation: n=7; dose-expansion, n=26). No dose-limiting toxicities were observed, and RP2D was established as SHR2554 350mg bid plus SHR-1701 30mg/kg q3w. Treatment-related adverse events (TRAEs) occurred in 13 of 32 patients (40.6%), mainly including platelet count decreased (21.9%), anemia (15.6%) and vomiting (15.6%). The most common Grade ≥3 TRAEs were anemia (3.1%) and fever (3.1%). In the efficacy-evaluable population (n = 26), objective response rate (ORR) was 57.7%, including 2 complete response (CR) achieved by patients with Metastatic Renal Cell Carcinoma and classical Hodgkin lymphoma (cHL). 16 patients with relapsed/refractory classical Hodgkin lymphoma (cHL) after a median of 10 prior lines of therapy have been enrolled. 16 (100%) cHL pts had received prior anti-PD1/PD-L1 antibody therapy, and 15 ( 93.8%) prior epigenetic therapies, such as decitabine or chidamide. Out of 14 cHL-evaluable patients, ORR was 100% and the CR rate was 7.1%. Conclusions: SHR-1701 combined with SHR2554 showed acceptable safety profile and encouraging antitumor activity in immunotherapy-pretreated cHL, establishing the foundation for further exploration. Clinical trial information: NCT04407741 .

Oxidative stress in the tumor microenvironment in gastric cancer and its potential role in immunotherapy.

Gastric cancer (GC) is the fourth leading cause of cancer-related death and the fifth most common malignant tumor globally. However, the clinical efficacy of conventional therapies is limited. Currently, immunotherapy is considered an effective therapeutic strategy for the management of various cancers, especially GC, but is of only limited benefit for GC patients. Accumulating evidence has revealed that oxidative stress plays a critical role in the regulation of immune responses within the tumor microenvironment (TME), affecting the efficacy of immunotherapies. Reactive oxygen species exert critical roles in enhancing antigen presentation, regulating immune responses, and preventing immunoescape. In this review, we summarize the dominant cancer immunotherapeutic strategies and describe the interaction between oxidative stress and the immune TME. We emphasize the underlying mechanisms of the efficacy of cancer immunotherapy, which involves its effects on oxidative stress, in the context of GC. We also highlight the therapeutic potential of regulating oxidative stress to improve immunotherapies, which may have benefits for clinical practice.

Bioengineered Nanospores Selectively Blocking LC3-Associated Phagocytosis in Tumor-Associated Macrophages Potentiate Antitumor Immunity.

Although cytotoxic treatments hold tremendous potential in boosting antitumor immunity, efferocytosis of tumor-associated macrophages (TAMs) could negatively remove apoptotic tumor cells through LC3-associated phagocytosis (LAP), resulting in inefficient tumor antigen presentation and immunosuppressive tumor microenvironment. To address this issue, we developed TAM-targeting nanospores (PC-CW) inspired by the predominant tropism of Rhizopus oryzae toward macrophages. To construct PC-CW, we disguised poly(sodium-p-styrenesulfonate) (PSS)-coated polyethylenimine (PEI)-shRNA nanocomplexes with the cell wall of R. oryzae conidia. LAP blockade by PC-CW delayed the degradation of engulfed tumor debris within TAMs, which not only enhanced antigen presentation but also initiated the domino effect of the antitumor immune response through STING signaling and TAM repolarization. Benefiting from this, PC-CW successfully sensitized the immune microenvironment and amplified CD8+ T cell responses following chemo-photothermal therapy, leading to substantial tumor growth control and metastasis prevention in tumor-bearing mouse models. The bioengineered nanospores represent a simple and versatile immunomodulatory strategy targeting TAMs for robust antitumor immunotherapy.

Prognostic association of immunoproteasome expression in solid tumours is governed by the immediate immune environment.

Induction of immunoproteasome (IP) expression in tumour cells can enhance antigen presentation and immunogenicity. Recently, overexpression of IP genes has been associated with better prognosis and response to immune checkpoint blockade (ICB) therapies in melanoma. However, the extent of this association in other solid tumours and how that is influenced by tumour cell-intrinsic and cell-extrinsic factors remains unclear. Here, we address this by exploring the gene expression patterns from available bulk and single-cell transcriptomic data of primary tumours. We find that tumours with high IP expression exhibit cytotoxic immune cell infiltration and upregulation of IFN-γ and TNF-α pathways in tumour cells. However, the association of IP expression with overall survival (TCGA cohort) and response to ICB therapy (non-TCGA cohorts) is tumour-type specific (better in non-small-cell lung, breast, bladder, and thymus; and worse in glioma and renal) and is greatly influenced by pro- or anti-tumorigenic immune cell infiltration patterns. This emphasises the need for considering immune cell infiltration patterns, along with IP expression, as a prognostic biomarker to predict overall survival or response to ICB therapies in solid tumours, besides melanoma.

Naïve T cell activation in the draining lymph nodes is accelerated by airway resident CD4 T cell activation

Abstract Tissue-resident memory CD4 T cells (TRM) protect against pathogens through the early shaping of inflammation at sites of infection. The extent to which their activation can impact subsequent systemic T cell responses is not clear. To ask if lung CD4 TRM recognizing antigen can impact systemic T cell responses against the same antigen presented in secondary lymphoid organs, we generated OVA-specific lung TRM primed by infection with an influenza virus expressing OVA peptide. We isolated the OVA-specific TRM 30 days after infection and transferred one million to naive mice intranasally resulting in an airway-resident TRM cohort similar in magnitude to TRM generated in virus-primed mice. We then recalled the transferred TRM by intranasal administration of FITC-labelled OVA protein to focus on TRM recall in the absence of virus-induced inflammation. One day after OVA challenge more FITC+ APC were present in the draining lymph nodes (dLN) of mice with TRM, suggesting antigen presentation to systemic T cells may be accelerated in the presence of airway TRM. We confirmed this by tracking naive OVA-specific CD4 T cells (OT-II) and CD8 T cell (OT-I) cohorts transferred i.v. to mice harboring OVA-specific TRM or not: more responding naive cells, expressing higher levels of activation markers, were detected 3 days after OVA challenge in mice with TRM. By 4 days, more responding cells were present in the lungs of TRM recipients, indicating that TRM and systemic T cell responses form a circuit via enhanced antigen presentation in the lymph nodes. Our results suggest that enhanced kinetics of systemic T cell activation induced by TRM activation in the airways may represent a novel mechanism of their protection against influenza and other respiratory viruses.

Abstract A28: Mechanisms of adaptive immune evasion by pre-leukemic DNMT3A-mutated blood cell clones

Abstract Hematopoietic stem cells (HSC) frequently acquire gene mutations with age. An expansion of blood cells derived from a single HSC is termed clonal hematopoiesis (CH), a condition that often precedes acute myeloid leukemia (AML). CH is common in elderly individuals: by age 60, approximately 10% of individuals have a clonally expanded blood cell population with a leukemia-associated mutation. The most common mutations in CH (50% of all cases) inactivate the DNA methyltransferase DNMT3A, which regulates gene expression via methylation of CpG rich regions. These mutations provide a selective advantage over healthy HSCs during aging and inflammation. Our goal is to discover mechanisms that allow for clonal expansion of DNMT3A-driven CH. A better understanding of these mechanisms will inspire new therapeutic strategies to limit clonal expansion and increase survival. To evaluate the transcriptional consequences of DNMT3A mutations in myeloid cells, we analyzed our previously published single-cell RNA-sequencing data of DNMT3A-mutated AML patient samples. We found a significant enrichment of antigen presentation signatures in DNMT3A mutated cells along the myeloid differentiation trajectory. CD8+ T cells from these same patients had significantly increased signatures of T cell activation and T cell exhaustion. T cell exhaustion occurs after an activated T cell has been continuously exposed to antigens. Exhausted T cells lack most effector functions and the ability to properly surveil (i.e. recognize mutant peptides presented on the surface of mutated cells and kill the target upon recognition) and restrain mutant cells from expanding. We hypothesize that DNMT3A-driven CH leads to escape of immune surveillance via chronic antigen stimulation and eventual promotion of T cell exhaustion. This would allow for further expansion of the mutated cells and the transformation of CH into AML. To functionally test this hypothesis, we evaluated whether a mouse model of Dnmt3a-CH recapitulated our human data. Indeed, scRNA-seq data of mouse Dnmt3a-CH cells showed significantly increased antigen presentation signatures across the myeloid differentiation trajectory. Additionally, CD8+ T cells had increased signatures of activation. We next functionally tested whether Dnmt3a-mutated CH cells had enhanced antigen presentation by pulsing bone marrow cells derived from Dnmt3a-CH mice with DQ-Ovalbumin, a self-quenched exogenous protein that becomes fluorescent when endocytosed and digested in the phagolysosome. Dnmt3a-mutated HSCs and LSKs had significantly increased DQ-OVA uptake and processing. In conclusion, we found that DNMT3A-CH cells have increased antigen presentation and processing which corresponds with enhanced immunogenicity. These findings support a model in which T cells constrain the expansion of DNMT3A-mutated HSCs, until T cell exhaustion occurs, leading to further expansion of the mutated clone and an increased chance of transformation into AML. Citation Format: Kyle A Romine, Daniel Ssozi, Tina Mujica, Yoke Lee, Jennifer Trowbridge, Peter van Galen. Mechanisms of adaptive immune evasion by pre-leukemic DNMT3A-mutated blood cell clones [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A28.

Adjuvant effects of peptidoglycan on CD4 +T cell activation by MHC-II +fibroblast-like synoviocytes

Abstract Lyme arthritis (LA) is the predominant late-stage manifestation of Lyme disease, caused by tick-borne spirochete Borrelia burgdorferi. LA can resolve with antibiotic therapy or persist after treatment in a subset of patients, known as post-antibiotic LA. Post-antibiotic LA is characterized by proliferative synovitis, dysregulated Th1/ IFNɣ responses and MHC-II+ fibroblast-like synoviocytes (FLS), a common cell type in inflamed joints. High levels of B. burgdorferi peptidoglycan (PG) have been found in LA patient synovia, yet the effects of PG on host immune responses are not well characterized. We hypothesize that PG acts as an adjuvant to enhance antigen presentation and CD4 +T cell activation in the joint. To test this, primary murine macrophages and FLS, both abundant in LA synovia, were treated in vitro with IFNɣ, PG, or both. Stimulation of macrophages led to upregulation of MHC-II in an IFNɣ-dependent manner, as expected. FLS stimulated with IFNɣ led to upregulation of MHC-II and production of proinflammatory cytokines, which was enhanced by PG as well as the muramyl-dipeptide subunit of PG, a known NOD2 ligand. To examine T cell responses, IFNɣ-primed MHC-II +FLS were co-cultured with OT-II mouse T cells (all with identical antigen-specific T cell receptors) and supplemented with the cognate OVA 323–339peptide. MHC-II +FLS activated naïve CD4 +T cell proliferation, which was enhanced by PG stimulation. Co-culture supernatants showed upregulated IFNɣ and IL-12 production, indicating a Th1 response. Negative controls using an alternative OVA 257–264peptide or NOD2-deficient FLS failed to induce T cell responses. These data show that PG acts as an immune adjuvant enhancing peptide-specific CD4 +T cell activation by MHC-II +FLS.

Combination adjuvants and antigens provide heterosubtypic protection against lethal influenza infection.

Abstract Influenza A virus (IAV) is a negative strand RNA virus that undergoes antigenic drift and shift, leading to serologically distinct strains that can cause tens of thousands of deaths annually in the US. The wide variety of hemagglutinin (HA) and neuraminidase (NA) proteins necessitates yearly reformulation of the vaccine to protect against novel seasonal and pandemic strains. Current IAV vaccines do not contain adjuvants, however, immune modulators such as pattern recognition receptor (PRR) agonists may enhance antigen presentation to T cells and provide broader protection against IAV strains that differ in HA and NA outer coat proteins. We set out to investigate whether TLR4 (lipopolysaccharide) and TLR3 (poly IC) agonists in combination with HA (H3N2) and nucleoprotein (NP) or NP and matrix (M1) proteins could provide protection against lethal challenge with mismatched IAV strain H1N1. Mice given H3 and NP with LPS + pIC in a prime/boost strategy demonstrated 80% survival after heterosubtypic H1N1 challenge while H3 + NP in PBS showed 20% survival. Similar results were seen using LPS + pIC in combination with NP and M1 protein with 90% survival. High endpoint titers of anti-NP antibodies (that also bound H1N1 virus) correlated with heterosubtypic protection suggesting a role for non-neutralizing antibodies in promoting survival. In addition, LPS and pIC enhanced dendritic cell (DC) function in vitro and increased migratory monocyte DC in the lymph node in vivo, coupled with early upregulation of T cell marker CD69. This strategy may provide increased efficacy over current IAV vaccines by inducing non-neutralizing antibodies and enhanced T cell responses to internal proteins. Supported by Trudeau Institute and Clarkson University.

Abstract A14: Ex vivo immune activation with the macrophage-targeting immunotherapy, anti-Clever-1 antibody bexmarilimab, in acute myeloid leukemia and myelodysplastic syndrome

Abstract The potential of immunotherapies in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remains still under investigation. Clever-1 (also known as Stabilin-1) is a multifunctional scavenger and adhesion receptor expressed by monocytes and immunosuppressive macrophages. Bexmarilimab, a Clever-1 targeting antibody, demonstrates many immunomodulatory effects1 along with promising anti-tumor activity against solid tumors in patients with multiple lines of previous treatment2. In AML, high STAB1 (mRNA) levels associate with poor survival and resistance to therapy3. The aim of this study was to profile Clever-1 expression in AML and MDS and test in preclinical models the growth inhibitory and immunomodulatory potential of bexmarilimab, as a single agent and in combination with azaciditine and venetoclax. AML cell lines (n=11) and frozen mononuclear cells, extracted from AML (n=42) and very high risk MDS (n=4) patient bone marrow (BM) aspirates provided by the Finnish Hematology Registry and Biobank, were used. Samples were treated for 48h with bexmarilimab alone, or in combination with azacytidine and/or venetoclax. Flow cytometry was used to detect different myeloid and TBNK cell populations along with Clever-1, HLA-DR, PD-(L)1 and additional T cell activation markers. Our results confirmed Clever-1 protein expression in AML cell lines and bexmarilimab treatment induced metabolic and growth inhibition of KG1 blasts (Clever-1high). In patients with AML, FAB M4/M5 subtypes exhibited highest Clever-1 levels, along with FAB M2 patients with consequent rapid relapse. Clever-1 expression correlated negatively with monocyte MHC class II molecule, HLA-DR, expression, and BM T-cell frequency, in line with the immunosuppressed state associated with high Clever-1. Ex vivo treatment of the primary AML BM cells with bexmarilimab resulted in a notable, 5-10x fold increase in monocyte HLA-DR in samples with low basal HLA-DR and high Clever-1. The combination of azacitidine with bexmarilimab augmented HLA-DR induction by 20-110% (mean 44%). FAB M1/M2 AML showed increase in activation markers, such as Ki67, CXCR3 and Granzyme B after ex vivo bexmarilimab treatment in CD8+ T-cells. Furthermore, bexmarilimab reduced PD-1 expression in NK- and CD8+CXCR3+ T-cell populations of FAB M0-M2 AML and MDS-EB2. This project is the first comprehensive investigation of Clever-1 expression in AML and MDS patient BM blasts and monocytes. Ex vivo treatment with bexmarilimab, alone or in combination with azacytidine or venetoclax, indicate enhanced antigen presentation capability and immunological activation. These results validate the therapeutic potential of bexmarilimab in myeloid malignancies. The safety, tolerability and preliminary efficacy of bexmarilimab is now further investigated in combination with venetoclax and/or azacytidine in a phase I/II clinical trial BEXMAB (NCT05428969).1Viitala et al., Clin Cancer Res 2019;25:3289-303; 2 Bono et al., Annals of Oncology; 2021. p 32, 5: S1283-S346; 3 Lin et al., Mol Ther Nucleic Acids 2019;18:476-84 Citation Format: Arno Ylitalo, Sofia Aakko, Heikki Kuusanmäki, Mari Björkman, Juho Jalkanen, Marie-Louise Fjällskog, Caroline Heckman, Maija Hollmén, Mika Kontro. Ex vivo immune activation with the macrophage-targeting immunotherapy, anti-Clever-1 antibody bexmarilimab, in acute myeloid leukemia and myelodysplastic syndrome [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A14.

Interleukin-17A knockout or self-recovery alleviated autoimmune reaction induced by fluoride in mouse testis

Fluoride (F) is usually treated as a hazardous material, and F-caused public health problem has attracted global attention. Previous studies demonstrate that interleukin-17A (IL-17A) plays a crucial role in F-elicited autoimmune orchitis and self-recovery reverses F-induced testicular toxicity to some extent, but these basic mechanisms remain unclear. Thus, we established a 180 d F exposure model of wild type (WT) mice and IL-17A knockout mice (C57BL/6 J background), and 60 d & 120 d self-recovery model based on F exposure model of WT mice, and used various techniques like qRT-PCR, western blot, immunohistochemistry and ELISA to further explore the mechanism of F-induced autoimmune reaction, the role of IL-17A in it and the reversibility of F-caused toxicity in testis. The results indicated that F exposure for 180 d caused the decreased sperm quality, the damaged testis histopathology, the enhanced mRNA and protein expression levels of inflammatory cytokines, the changes of autoantibody such as the appearance and increased content of anti-testicular autoantibodies in sera and the autoantibody deposition in testis, the alterations of autoimmune related genes containing the decreased mRNA and protein expressions of AIRE and FOXP3 with an increase of MHCII, and the reduced protein expressions of CTLA4, and the activation of IL-17A signaling cascade like the elevated mRNA and protein expressions of IL-17A, Act1, NF-κB, AP-1 and CEBPβ, and the increased protein expressions of IL-17RC, with a decrease of IκBα. After IL-17A knockout, 29 of 35 F-induced changes were alleviated. In two self-recovery models, all F-caused differences except fluorine concentration in femur were gradually restored in a time-dependent manner. This study concluded that IL-17A knockout or self-recovery attenuated F-induced testicular injury and decrease of sperm quality through alleviating autoimmune reaction which was involved with the activation of IL-17A pathway, the damage of self-tolerance and the enhancement of antigen presentation.

Abstract LB279: Targeting microglial metabolic rewiring synergizes with immune checkpoint blockade therapy for glioblastoma

Abstract Glioblastoma (GBM) constitutes the most lethal primary brain tumor for which immunotherapy has provided limited benefit. The unique brain immune landscape is reflected in a complex tumor immune microenvironment (TIME) in GBM. Here, single cell sequencing of the GBM TIME revealed that microglia were under severe oxidative stress, which induced nuclear receptor subfamily 4 group A member 2 (NR4A2)-dependent transcriptional activity in microglia. Heterozygous Nr4a2 (Nr4a2 +/−) or microglia-specific Nr4a2 (Nr4a2 fl/fl Cx3cr1 cre) genetic targeting reshaped microglia plasticity in vivo by reducing alternatively activated microglia and enhancing antigen presentation capacity for CD8+ T cells in GBM. In microglia, NR4A2 activated squalene monooxygenase (SQLE) to dysregulate cholesterol homeostasis. Pharmacological NR4A2 inhibition attenuated the pro-tumorigenic TIME, and targeting the NR4A2 or SQLE enhanced therapeutic efficacy of immune checkpoint blockade in vivo. Collectively, oxidative stress promotes tumor growth through NR4A2-SQLE activity in microglia, informing novel immune therapy paradigms in brain cancer. Citation Format: Zengpanpan Ye, Xiaolin Ai, Linjie Zhao, Jeremy N. Rich, Shengtao Zhou. Targeting microglial metabolic rewiring synergizes with immune checkpoint blockade therapy for glioblastoma [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 LB279.

Abstract 6384: ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression

Abstract Background: Dendritic cells (DCs) are vital for initiating antigen-specific T cell-dependent antitumor immune responses. However, emerging evidence reveals that local DCs within the tumor microenvironment (TME) are tolerized to facilitate immune evasion. Reprogramming of DCs is recognized as a promising strategy for tumor immunotherapy. Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor belonging to the LILR family which is mainly expressed on normal myeloid cells and myeloid-derived malignant cells. It is upregulated on tolerogenic DCs (tolDCs), which exhibit low expression levels of costimulatory molecules and resistance to DC maturation. Targeting LILRB4 to reprogram tolDCs has been reported to be a promising strategy for cancer treatment. Here we report the preclinical development of an LILRB4 antagonist antibody, ATG-034. Method: The protein-based and cell-based binding affinity of ATG-034 were measured using SPR, ELISA and FACS analysis. Fc receptor (FcR) stimulation assay was used to evaluate the ability of ATG-034 to block the interaction of LILRB4 with its ligand fibronectin. ATG-034-mediated enhancement of antigen presentation and co-stimulation ability of DCs was determined by FACS analysis for the change of surface expression of HLA-DR, HLA-ABC, CD86, and CD206. The mixed lymphocyte reaction (MLR) assay was used to assess the immunomodulatory potential of ATG-034. The in vivo antitumor efficacy of ATG-034 was evaluated in a radiation therapy-resistant murine Lewis lung carcinoma (LLC) syngeneic model. Results: ATG-034 binds to LILRB4 protein with a single-digit nM affinity. It potently reversed the fibronectin-mediated inhibition of FcR-driven TNF-α cytokine production. TolDCs were reprogrammed by ATG-034 to immunogenic DCs with significant upregulation of the expression of HLA-DR, HLA-ABC, CD86 and downregulation of the expression of CD206. Furthermore, ATG-034 remarkably reinvigorated tolDCs for priming T cell activation with noticeable upregulation of the expression of CD25 and the secretion of IFN-γ, enhancing antitumor immunity. In addition, 10 mg/kg ATG-034 significantly inhibited the LLC tumor growth in vivo with a TGI of 40.28%, while a benchmark antibody only demonstrated a 24.43% TGI. Conclusion: Our data show that ATG-034 reprograms tolDC, enhancing anti-tumor immunity, and demonstrates potent in vivo anti-tumor efficacy. Therefore, ATG-034 may be a promising strategy for the treatment of cancer. Citation Format: Ao Sun, Enlin Zheng, Huili Cao, Mengshi Cao, Suya Bai, Peng Chen, Linjie Tian, Jay Mei, Bo Shan, Bing Hou. ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6384.