In vitro antibody-mediated SARS-CoV-2 infection suppression through human ACE2 receptor blockade.

Vaccines and antibodies that specifically target or neutralize components of the SARS-CoV-2 virus are effective in prevention and treatment of human patients with SARS-CoV-2 infection. However, vaccines and SARS-CoV-2 neutralization antibodies target a subset of epitopes of viral proteins, and the fast evolution of the SARS-CoV-2 virus and the continuing emergence of SARS-CoV-2 variants confer SARS-CoV-2 immune escape from these therapies. ACE2 is the human cell receptor that serves as the entry point for SARS-CoV-2 into human cells and thus is the gatekeeper for SARS-CoV-2 infection of humans. We report here the development of 4G8C11, an anti-human ACE2 receptor monoclonal antibody that recognizes ACE2 on human cell surfaces. We determined that 4G8C11 blocks SARS-CoV-2 and variant infection of ACE2+ human cells. Furthermore, 4G8C11 has minimal effects on ACE2 receptor activity. 4G8C11 is therefore a monoclonal antibody for ACE2 receptor detection and potentially an effective immunotherapeutic agent for SARS-CoV-2 and variants.

Tumor PD-L1 engages myeloid PD-1 to suppress type I interferon to impair cytotoxic T lymphocyte recruitment.

The cellular and molecular mechanisms underlying tumor cell PD-L1 (tPD-L1) function in tumor immune evasion are incompletely understood. We report here that tPD-L1 does not suppress cytotoxic T lymphocyte (CTL) activity in co-cultures of tumor cells and tumor-specific CTLs and exhibits no effect on primary tumor growth. However, deleting tPD-L1 decreases lung metastasis in a CTL-dependent manner in tumor-bearing mice. Depletion of myeloid cells or knocking out PD-1 in myeloid cells (mPD-1) impairs tPD-L1 promotion of tumor lung metastasis in mice. Single-cell RNA sequencing (scRNA-seq) reveals that tPD-L1 engages mPD-1 to activate SHP2 to antagonize the type I interferon (IFN-I) and STAT1 pathway to repress Cxcl9 and impair CTL recruitment to lung metastases. Human cancer patient response to PD-1 blockade immunotherapy correlates with IFN-I response in myeloid cells. Our findings determine that tPD-L1 engages mPD-1 to activate SHP2 to suppress the IFN-I-STAT1-CXCL9 pathway to impair CTL tumor recruitment in lung metastasis.

IRF8 Regulates Intrinsic Ferroptosis through Repressing p53 Expression to Maintain Tumor Cell Sensitivity to Cytotoxic T Lymphocytes.

Ferroptosis has emerged as a cytotoxic T lymphocyte (CTL)-induced tumor cell death pathway. The regulation of tumor cell sensitivity to ferroptosis is incompletely understood. Here, we report that interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified the ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells acquire resistance to intrinsic ferroptosis induction and IRF8-deficient tumor cells also exhibit decreased ferroptosis in response to tumor-specific CTLs. Irf8 deletion increased p53 expression in tumor cells and knocking out p53 in IRF8.KO tumor cells restored tumor cell sensitivity to intrinsic ferroptosis induction. Furthermore, IRF8.KO tumor cells grew significantly faster than WT tumor cells in immune-competent mice. To restore IRF8 expression in tumor cells, we designed and synthesized codon usage-optimized IRF8-encoding DNA to generate IRF8-encoding plasmid NTC9385R-mIRF8. Restoring IRF8 expression via a lipid nanoparticle-encapsulated NTC9385R-mIRF8 plasmid therapy suppressed established tumor growth in vivo. In human cancer patients, nivolumab responders have a significantly higher IRF8 expression level in their tumor cells as compared to the non-responders. Our data determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis.

IRF8: Mechanism of Action and Health Implications.

Interferon regulatory factor 8 (IRF8) is a transcription factor of the IRF protein family. IRF8 was originally identified as an essentialfactor for myeloid cell lineage commitment and differentiation. Deletion of Irf8 leads to massive accumulation of CD11b+Gr1+ immature myeloid cells (IMCs), particularly the CD11b+Ly6Chi/+Ly6G- polymorphonuclear myeloid-derived suppressor cell-like cells (PMN-MDSCs). Under pathological conditions such as cancer, Irf8 is silenced by its promoter DNA hypermethylation, resulting in accumulation of PMN-MDSCs and CD11b+ Ly6G+Ly6Clo monocytic MDSCs (M-MDSCs) in mice. IRF8 is often silenced in MDSCs in human cancer patients. MDSCs are heterogeneous populations of immune suppressive cells that suppress T and NK cell activity to promote tumor immune evasion and produce growth factors to exert direct tumor-promoting activity. Emerging experimental data reveals that IRF8 is also expressed in non-hematopoietic cells. Epithelial cell-expressed IRF8 regulates apoptosis and represses Osteopontin (OPN). Human tumor cells may use the IRF8 promoter DNA methylation as a mechanism to repress IRF8 expression to advance cancer through acquiring apoptosis resistance and OPN up-regulation. Elevated OPN engages CD44 to suppress T cell activation and promote tumor cell stemness to advance cancer. IRF8 thus is a transcription factor that regulates both the immune and non-immune components in human health and diseases.

Lipid Nanoparticle Delivery of Fas Plasmid Restores Fas Expression to Suppress Melanoma Growth In Vivo.

Fas ligand (FasL), expressed on the surface of activated cytotoxic T lymphocytes (CTLs), is the physiological ligand for the cell surface death receptor, Fas. The Fas-FasL engagement initiates diverse signaling pathways, including the extrinsic cell death signaling pathway, which is one of the effector mechanisms that CTLs use to kill tumor cells. Emerging clinical and experimental data indicate that Fas is essential for the efficacy of CAR-T cell immunotherapy. Furthermore, loss of Fas expression is a hallmark of human melanoma. We hypothesize that restoring Fas expression in tumor cells reverses human melanoma resistance to T cell cytotoxicity. DNA hypermethylation, at the FAS promoter, down-regulates FAS expression and confers melanoma cell resistance to FasL-induced cell death. Forced expression of Fas in tumor cells overcomes melanoma resistance to FasL-induced cell death in vitro. Lipid nanoparticle-encapsulated mouse Fas-encoding plasmid therapy eliminates Fas+ tumor cells and suppresses established melanoma growth in immune-competent syngeneic mice. Similarly, lipid nanoparticle-encapsulated human FAS-encoding plasmid (hCOFAS01) therapy significantly increases Fas protein levels on tumor cells of human melanoma patient-derived xenograft (PDX) and suppresses the established human melanoma PDX growth in humanized NSG mice. In human melanoma patients, FasL is expressed in activated and exhausted T cells, Fas mRNA level positively correlates with melanoma patient survival, and nivolumab immunotherapy increases FAS expression in tumor cells. Our data demonstrate that hCOFAS01 is an effective immunotherapeutic agent for human melanoma therapy with dual efficacy in increasing tumor cell FAS expression and in enhancing CTL tumor infiltration.

Restoring FAS Expression via Lipid-Encapsulated FAS DNA Nanoparticle Delivery Is Sufficient to Suppress Colon Tumor Growth In Vivo.

A hallmark of human colorectal cancer is lost expression of FAS, the death receptor for FASL of cytotoxic T lymphocytes (CTLs). However, it is unknown whether restoring FAS expression alone is sufficient to suppress csolorectal-cancer development. The FAS promoter is hypermethylated and inversely correlated with FAS mRNA level in human colorectal carcinomas. Analysis of single-cell RNA-Seq datasets revealed that FAS is highly expressed in epithelial cells and immune cells but down-regulated in colon-tumor cells in human colorectal-cancer patients. Codon usage-optimized mouse and human FAS cDNA was designed, synthesized, and encapsulated into cationic lipid to formulate nanoparticle DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Overexpression of codon usage-optimized FAS in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro, suppressed colon tumor growth, and increased the survival of tumor-bearing mice in vivo. Overexpression of codon-optimized FAS-induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. DOTAP-Chol-mFAS therapy exhibited no significant liver toxicity. Our data determined that tumor-selective delivery of FAS DNA nanoparticles is sufficient for suppression of human colon tumor growth in vivo.

WDR5-H3K4me3 epigenetic axis regulates OPN expression to compensate PD-L1 function to promote pancreatic cancer immune escape.

BACKGROUND: Despite PD-L1 (Programmed death receptor ligand-1) expression on tumor cells and cytotoxic T lymphocytes tumor infiltration in the tumor microenvironment, human pancreatic cancer stands out as one of the human cancers that does not respond to immune checkpoint inhibitor (ICI) immunotherapy. Epigenome dysregulation has emerged as a major mechanism in T cell exhaustion and non-response to ICI immunotherapy, we, therefore, aimed at testing the hypothesis that an epigenetic mechanism compensates PD-L1 function to render pancreatic cancer non-response to ICI immunotherapy. METHODS: Two orthotopic pancreatic tumor mouse models were used for chromatin immunoprecipitation-Seq and RNA-Seq to identify genome-wide dysregulation of H3K4me3 and gene expression. Human pancreatic tumor and serum were analyzed for osteopontin (OPN) protein level and for correlation with patient prognosis. OPN and PD-L1 cellular location were determined in the tumors using flow cytometry. The function of WDR5-H3K4me3 axis in OPN expression were determined by Western blotting. The function of H3K4me3-OPN axis in pancreatic cancer immune escape and response to ICI immunotherapy was determined in an orthotopic pancreatic tumor mouse model. RESULTS: Mouse pancreatic tumors have a genome-wide increase in H3K4me3 deposition as compared with normal pancreas. OPN and its receptor CD44 were identified being upregulated in pancreatic tumors by their promoter H3K4me3 deposition. OPN protein is increased in both tumor cells and tumor-infiltrating immune cells in human pancreatic carcinoma and is inversely correlated with pancreatic cancer patient survival. OPN is primarily expressed in tumor cells and monocytic myeloid-derived suppressor cells (M-MDSCs), whereas PD-L1 is expressed in tumor cells, M-MDSCs, polymorphonuclear MDSCs and tumor-associated macrophages. WDR5 is essential for H3K4me3-specific histone methyltransferase activity that regulates OPN expression in tumor cells and MDSCs. Inhibition of WDR5 significantly decreased OPN protein level. Inhibition of WDR5 or knocking out of OPN suppressed orthotopic mouse pancreatic tumor growth. Inhibition of WDR5 also significantly increased efficacy of anti-PD-1 immunotherapy in suppression of mouse pancreatic tumor growth in vivo. CONCLUSIONS: OPN compensates PD-L1 function to promote pancreatic cancer immune escape. Pharmacological inhibition of the WDR5-H3K4me3 epigenetic axis is effective in suppressing pancreatic tumor immune escape and in improving efficacy of anti-PD-1 immunotherapy in pancreatic cancer.

Osteopontin Blockade Immunotherapy Increases Cytotoxic T Lymphocyte Lytic Activity and Suppresses Colon Tumor Progression.

Human colorectal cancers are mostly microsatellite-stable with no response to anti-PD-1 blockade immunotherapy, necessitating the development of a new immunotherapy. Osteopontin (OPN) is elevated in human colorectal cancer and may function as an immune checkpoint. We aimed at elucidating the mechanism of action of OPN and determining the efficacy of OPN blockade immunotherapy in suppression of colon cancer. We report here that OPN is primarily expressed in tumor cells, myeloid cells, and innate lymphoid cells in human colorectal carcinoma. Spp1 knock out mice exhibit a high incidence and fast growth rate of carcinogen-induced tumors. Knocking out Spp1 in colon tumor cells increased tumor-specific CTL cytotoxicity in vitro and resulted in decreased tumor growth in vivo. The OPN protein level is elevated in the peripheral blood of tumor-bearing mice. We developed four OPN neutralization monoclonal antibodies based on their efficacy in blocking OPN inhibition of T cell activation. OPN clones 100D3 and 103D6 increased the efficacy of tumor-specific CTLs in killing colon tumor cells in vitro and suppressed colon tumor growth in tumor-bearing mice in vivo. Our data indicate that OPN blockade immunotherapy with 100D3 and 103D6 has great potential to be further developed for colorectal cancer immunotherapy and for rendering a colorectal cancer response to anti-PD-1 immunotherapy.

Asah2 Represses the p53-Hmox1 Axis to Protect Myeloid-Derived Suppressor Cells from Ferroptosis.

Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC50 of 10.16-25.91 µM for human ASAH2 and 18.6-30.2 µM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione synthesis and decreases lipid reactive oxygen species to suppress ferroptosis in MDSCs. Gene expression profiling identified the p53 pathway as the Asah2 target in MDSCs. Inhibition of Asah2 increased p53 protein stability to upregulate Hmox1 expression to suppress lipid reactive oxygen species production to suppress ferroptosis in MDSCs. NC06 therapy increases MDSC death and reduces MDSC accumulation in tumor-bearing mice, resulting in increased activation of tumor-infiltrating CTLs and suppression of tumor growth in vivo. Our data indicate that ASAH2 protects MDSCs from ferroptosis through destabilizing p53 protein to suppress the p53 pathway in MDSCs in the tumor microenvironment. Targeting ASAH2 with NC06 to induce MDSC ferroptosis is potentially an effective therapy to suppress MDSC accumulation in cancer immunotherapy.

Osteopontin: A Key Regulator of Tumor Progression and Immunomodulation.

OPN is a multifunctional phosphoglycoprotein expressed in a wide range of cells, including osteoclasts, osteoblasts, neurons, epithelial cells, T, B, NK, NK T, myeloid, and innate lymphoid cells. OPN plays an important role in diverse biological processes and is implicated in multiple diseases such as cardiovascular, diabetes, kidney, proinflammatory, fibrosis, nephrolithiasis, wound healing, and cancer. In cancer patients, overexpressed OPN is often detected in the tumor microenvironment and elevated serum OPN level is correlated with poor prognosis. Initially identified in activated T cells and termed as early T cell activation gene, OPN links innate cells to adaptive cells in immune response to infection and cancer. Recent single cell RNA sequencing revealed that OPN is primarily expressed in tumor cells and tumor-infiltrating myeloid cells in human cancer patients. Emerging experimental data reveal a key role of OPN is tumor immune evasion through regulating macrophage polarization, recruitment, and inhibition of T cell activation in the tumor microenvironment. Therefore, in addition to its well-established direct tumor cell promotion function, OPN also acts as an immune checkpoint to negatively regulate T cell activation. The OPN protein level is highly elevated in peripheral blood of human cancer patients. OPN blockade immunotherapy with OPN neutralization monoclonal antibodies (mAbs) thus represents an attractive approach in human cancer immunotherapy.