An engineered mycobacterium smegmatis expressing anti-PD-L1/IL-15 immunocytokine induces and activates specific antitumor immunity
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PROVIDER: PRJNA1176547 | ENA |
REPOSITORIES: ENA
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Project description:Immune checkpoint inhibitors and cytokines have revolutionized tumor treatment but are still limited by dose-dependent toxicity and efficacy. In situ vaccine platforms based on intelligent microbes are promising therapeutic strategies, which can sustainably deliver drugs locally without causing severe systemic risks. Here, we have innovatively engineered a nonpathogenic, adjuvant-acting Mycobacterium smegmatis (M. smegmatis) that coexpresses a PD-L1 inhibitor and an IL-15 cytokine complex containing the IL-15Rα sushi domain (Ms-PDL1scfv-IL15). We demonstrate that PD-L1 inhibitor and IL-15 secretion systemically binds mouse or human PD-L1 and maintains IL-15 stimulatory activity. The bifunctional Ms-PDL1scfv-IL15 overcomes resistance to PD-L1 blockade, recruits numerous immune cells in situ, induces dendritic cells (DCs) maturation, initiates the M1 anti-tumor polarization of macrophages, increases the proliferation and activation of NK cells and tumor-infiltrating CD8+ T cells, inhibits the Tregs, elicits abscopal effects, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in several syngeneic tumor mouse models. We also found that the combination of Ms-PDL1scfv-IL15 with GM–CSF synergistically stunted the tumor progress and stasis. Moreover, intratumoral administration of Ms-PDL1scfv-IL15 can capture tumor antigen fragments, and boost DCs presentation of antigens, which remarkably initiates tumor antigen-specific immune response, leading to durable tumor regression and specific antitumor immunity. In summary, this engineered M. smegmatis to recruit and activate innate and adaptive antitumor immune responses, offering a potent cancer immunotherapy strategy to treat patients with cold tumors or resistance to checkpoint blockade.
2024-12-25 | GSE280146 | GEO
Project description:Epigenetic mechanism contributes to immune landscapes in cancer. Here we identify the SETDB1-TRIM28 complex as a critical suppressor of antitumor immunity. An epigenetic CRISPR-Cas9 screen of 1,218 chromatin regulators identified TRIM28 as a novel suppressor of PD-L1 expression. We revealed that expression of the SETDB1-TRIM28 complex negatively correlates with infiltration of effector CD8+ T cells. Inhibition of SETDB1-TRIM28 simultaneously upregulates PD-L1 and activates the cGAS-STING innate immune response to increase infiltration of CD8+ T cells. Mechanistically, SETDB1-TRIM28 inhibition leads to micronuclei formation in cytoplasm, a known activator of the cGAS-STING pathway. Thus, SETDB1-TRIM28 inhibition bridges the innate and adaptive immunity. Indeed, SETDB1 knockout enhances the antitumor effects of immune checkpoint blockade anti-PD-L1 in an ovarian cancer mouse model in a cGAS dependent manner. Our findings establish SETDB1-TRIM28 complex as a regulator of antitumor immunity and its loss activates cGAS-STING innate immunity to boost antitumor effects of immune checkpoint blockades.
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Project description:Epigenetic mechanism contributes to immune landscapes in cancer. Here we identify the SETDB1-TRIM28 complex as a critical suppressor of antitumor immunity. An epigenetic CRISPR-Cas9 screen of 1,218 chromatin regulators identified TRIM28 as a novel suppressor of PD-L1 expression. We revealed that expression of the SETDB1-TRIM28 complex negatively correlates with infiltration of effector CD8+ T cells. Inhibition of SETDB1-TRIM28 simultaneously upregulates PD-L1 and activates the cGAS-STING innate immune response to increase infiltration of CD8+ T cells. Mechanistically, SETDB1-TRIM28 inhibition leads to micronuclei formation in cytoplasm, a known activator of the cGAS-STING pathway. Thus, SETDB1-TRIM28 inhibition bridges the innate and adaptive immunity. Indeed, SETDB1 knockout enhances the antitumor effects of immune checkpoint blockade anti-PD-L1 in an ovarian cancer mouse model in a cGAS dependent manner. Our findings establish SETDB1-TRIM28 complex as a regulator of antitumor immunity and its loss activates cGAS-STING innate immunity to boost antitumor effects of immune checkpoint blockades.
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Project description:Adoptively transferred T cells and agents designed to block the CD47/SIRPalpha axis are promising cancer therapeutics that activate distinct arms of the immune system. We administered anti-CD47 with adoptively transferred T cells with the goal of enhancing antitumor efficacy but observed rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors, which abrogated therapeutic benefit. anti-CD47 mediated CAR T clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered a CD47 variant (47E) that engaged SIRPalpha and provided a “don’t-eat-me” signal that was not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E were resistant to clearance by macrophages following anti-CD47, and mediated significant, sustained macrophage recruitment into the TME. Although many of the recruited macrophages manifested an M2-like profile, the combined therapy synergistically enhanced antitumor efficacy. This work identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T cell directed therapeutics with those designed to activate macrophages. It further delivers a therapeutic approach capable of simultaneously harnessing the antitumor effects of T cells and macrophages that manifests enhanced potency against solid tumors.
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Project description:RNA-seq of Mycobacteriophage Island3 infection of Mycolicibacterium smegmatis mc2155, Mycolicibacterium smegmatis mc2155(Butters), and Mycolicibacterium smegmatis mc2155(Buttersgp57r) to assess the impact of Butters lysogen and specifically Buttersgp57r on transcript levels of island3 during infection.
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Project description:Achieving robust responses with adoptive cell therapy for the treatment of the highly lethal pancreatic ductal adenocarcinoma (PDA) has been elusive. We previously showed that T cells engineered to express a mesothelin-specific T cell receptor (TCR) accumulate in autochthonous PDA, mediate therapeutic antitumor activity, but fail to eradicate tumors in part due to acquisition of a dysfunctional exhausted T cell state. Here, we investigated the role of immune checkpoints in mediating TCR engineered T cell dysfunction in a genetically engineered PDA mouse model. The fate of engineered T cells that were either deficient in PD-1, or transferred concurrent with antibodies blocking PD-L1 and/or additional immune checkpoints, were tracked to evaluate persistence, functionality, and antitumor activity at day 8 and day 28 post infusion. We performed RNAseq on engineered T cells isolated from tumors and compared differentially expressed genes to prototypical endogenous exhausted T cells.
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Project description:Epigenetic regulators have emerged as exciting targets for cancer therapy. Additionally, restoration of antitumor immunity by blocking the PD-L1 signaling using antibodies has proven to be beneficial in cancer therapy. Here we show that BET bromodomain inhibition suppresses PD-L1 expression and restores antitumor immunity in ovarian cancer. CD274 (encoding PD-L1) is a direct target of BRD4-mediated gene transcription. In mouse models, treatment with the BET inhibitor JQ1 significantly reduced PD-L1 expression on tumor cells and tumor-associated dendritic cells and macrophages, which correlated with an increase in the activity of antitumor cytotoxic T cells. Together, these data demonstrate an epigenetic approach to block PD-L1 signaling to restore antitumor immunity. Given the fact that BET inhibitors have been proven safe with manageable reversible toxicity in clinical trials, our findings indicate that pharmacological BET inhibitors represent a novel treatment strategy for targeting PD-L1 expression.
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Project description:Abstract: Radiation therapy is a key component of the standard of care for glioblastoma (GBM). Although this treatment is known to trigger pro-inflammatory immune responses, it also results in several immune resistance mechanisms such as the upregulation of CD47 by tumors leading to avoidance of phagocytosis and the overexpression of PD-L1 in tumor-associated myeloid cells (TAMCs). Leveraging these RT-elicited processes, we generated a bispecific-lipid nanoparticle (B-LNP) that engaged TAMCs to glioma cells via anti-CD47/PD-L1 dual-ligation. We show that B-LNP blocked these two vital immune checkpoint molecules and promoted the phagocytic activity of TAMCs. In order to boost subsequent T cell recruitment and antitumor activity after tumor engulfment, the B-LNP was encapsulated with diABZI, a non-nucleotidyl agonist for stimulator of interferon genes (STING). In vivo treatment with the diABZI-loaded B-LNP induced a transcriptomic and metabolic switch in TAMCs, transforming them into potent antitumor effector cells, which induced T cell infiltration and activation of in the brain tumors. In preclinical murine glioma models, B-LNP therapy significantly potentiated the antitumor effects of radiotherapy, promoted brain tumor regression, and induced immunological memory against gliomas. The nano37 therapy was efficacious through both intra-tumoral and systemic delivery routes. In summary, our study shows a unique nanotechnology-based approach that hijacks multiple immune checkpoints to boost potent and long-lasting antitumor immunity against GBM.
2023-02-06 | ST002467 | MetabolomicsWorkbench
Project description:Mycobacterium smegmatis SigF is a group III sigma factor. Its ortholog in M. tuberculosis is reported to have role in regulation and function of cell wall components. In present study we have created an M. smegmatis ΔsigF mutant by allele exchange method. M. smegmatis sigF mutant shows non pigmented phenotype and is more sensitive to hydrogen peroxide generated oxidative stress. DNA microarray analysis of M. smegmatis wild type and ΔsigF mutant suggests that SigF in this species controls the expression of several energy and central intermediary metabolism genes along with regulation of carotenoid biosynthesis.
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Project description:Adoptive transfer of genetically or nanoparticle-engineered macrophages represents a promising cell therapy modality for treatment of solid tumor. However, the therapeutic efficacy is suboptimal without achieving a complete tumor regression, and the underlying mechanism remains elusive. Here, we discover a subpopulation of cancer cells with upregulated CD133 and programmed death-ligand 1 in mouse melanoma, resistant to the phagocytosis by the transferred macrophages. Compared to the CD133-PD-L1- cancer cells, the CD133+PD-L1+ cancer cells express higher transforming growth factor-β signaling molecules to foster a resistant tumor niche, that restricts the trafficking of the transferred macrophages by stiffened extracellular matrix, and inhibits their cell-killing capability by immunosuppressive factors. The CD133+PD-L1+ cancer cells exhibit tumorigenic potential. The CD133+PD-L1+ cells are further identified in the clinically metastatic melanoma. Hyperthermia reverses the resistance of CD133+PD-L1+ cancer cells through upregulating the ‘eat me’ signal calreticulin, significantly improving the efficacy of adoptive macrophage therapy. Our findings demonstrate the mechanism of resistance to adoptive macrophage therapy, and provide a de novo strategy to counteract the resistance.
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